Antiviral compounds and methods of use thereof

ABSTRACT

Inhibitors of retroviral propagation, methods of treatment and prevention of retroviral infections using the inhibitors, and pharmaceutical compositions including the inhibitors, are disclosed.

CROSS-REFERENCE

This Application is a divisional application claiming priority to U.S.Ser. No. 13/634,464, filed on Sep. 12, 2012, which in turn is thenational stage entry under 35 U.S.C. 371 of PCT/US11/28397, filed onMar. 14, 2011, which in turn claims priority to U.S. ProvisionalApplication No. 61/313,527, filed on Mar. 12, 2010, the contents of eachof which are hereby incorporated by reference for all purposes.

FIELD

The invention generally relates to antiviral compounds, compositionsincluding the compounds, and methods of treatment using the compounds.

BACKGROUND

The primate lentiviruses include the human immunodeficiency virusestypes 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency viruses(SIVs) (Barre-Sinoussi, F., et al. (1983) Science 220:868-871; Clavel,F. (1987) AIDS 1:135-140; Daniel, M. D., et al. (1985) Science228:1201-1204; Desrosiers, R. C. (1990) Ann. Rev. Immunol. 8: 557-578;Gallo, R. C, et al. (1984) Science 224:500-503). HIV-1 and HIV-2 infecthumans, HIV-1-like viruses infect chimpanzees, and SIV variants infectAfrican monkeys. Humans infected by HIV-1 and HIV-2 and Asian macaquesinfected by certain SIV strains often develop life-threateningimmunodeficiency due to depletion of CD4-positive T lymphocytes (Fauci,A., et al. (1984) Ann. Int. Med. 100:91-106; Letvin, N. L., et al.(1985) Science 230:71-739,19).

In humans, HIV infection causes Acquired Immunodeficiency Syndrome(AIDS), an incurable disease in which the body's immune system breaksdown leaving the victim vulnerable to opportunistic infections, e.g.,pneumonia and certain cancers, e.g., Kaposi's Sarcoma. AIDS is a majorglobal health problem. The Joint United Nations Programme on HIV/AIDS(UNAIDS) estimates that there are now over 34 million people living withHIV or AIDS worldwide; some 28.1 million of those infected individualsreside in impoverished subSaharan Africa. In the United States,approximately one out of every 500 people are infected with HIV or haveAIDS. Since the beginning of the epidemic, AIDS has killed nearly 19million people worldwide, including some 425,000 Americans. AIDS hasreplaced malaria and tuberculosis as the world's deadliest infectiousdisease among adults and is the fourth leading cause of death worldwide.

There remains a need for the identification of inhibitors of retroviralinfection.

SUMMARY

Compounds which are inhibitors of retroviral propagation are disclosed.Methods of treating and/or preventing retroviral infection using theinhibitors of retroviral propagation, and pharmaceutical compositionsincluding the inhibitors and a pharmaceutically-acceptable carrier, arealso disclosed. Combination therapy using one or more of the inhibitors,and a second anti-retroviral compound, are also disclosed.

The compounds inhibit retroviral propagation by inhibiting retroviralreverse transcription, viral recruitment of the retroviral primer usedin translation, human tRNA^(Lys3), inhibiting the final packaging andassembly of new virions, and/or inhibiting the binding of a host celltRNA to a target nucleic acid molecule.

The inhibitory activity of the compounds can be evaluated using methodsfor screening inhibitors of retroviral propagation. Such methods mayinvolve forming a mixture comprising a linear sequence of a tRNAanticodon stem loop fragment, a nucleic acid molecule capable of bindingto the tRNA anticodon stem loop fragment, and a test compound. Themixture is incubated under conditions that allow binding of the tRNAanticodon stem loop fragment and the nucleic acid molecule in theabsence of the test compound. One can then determine whether or not atest compound inhibits the propagation of a retrovirus. Inhibition ofbinding of the tRNA ASL fragment and the target nucleic acid molecule isindicative of the test compound being an inhibitor of retroviralpropagation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic representation of the protection of themodified nucleotides prior to synthesis of the RNA oligomer. Panel Aillustrates protection with trifluoryl acetic acid. Panel B illustratesprotection with benzoyl. Panel C illustrates the general protection ofthe ribose hydroxyl groups.

FIG. 2A provides a representation of a labeled tRNA fragment and acorresponding target sequence. FIG. 2B provides structures of severalrepresentative modified nucleosides.

FIG. 3 provides a comparison of one example of an immobilized assay andan assay using the AlphaScreen™ assay.

DETAILED DESCRIPTION

The present invention relates to compounds which inhibit retroviralpropogation, compositions including the compounds, and methods oftreating and/or preventing retroviral infection using the compounds.Viral propagation can be inhibited by inhibiting reverse transcription,viral replication, translation of viral RNA into proteins, recruitmentof human tRNA^(Lys3), packaging and assembly of new virions, and/orinhibiting the binding of a host cell tRNA to a target nucleic acidmolecule.

Prior to describing this invention in further detail, however, thefollowing terms will first be defined.

Definitions

As used herein, an “inhibitor” refers to any compound capable ofpreventing, reducing, or restricting retroviral propagation. Aninhibitor may inhibit retroviral propagation, for example, bypreventing, reducing or restricting retroviral reverse transcription. Insome embodiments, the inhibition is at least 20% (e.g., at least 50%,70%, 80%, 90%, 95%, 98%, 99%, 99.5%) of the retroviral propagation ascompared to the propagation in the absence of the inhibitor. In oneaspect, an inhibitor prevents, reduces, or restricts the binding of atRNA, or fragment thereof, to a target nucleic acid molecule. Inhibitorscan also affect recruitment of human tRNA^(Lys3), translation of viralRNA into proteins, and/or final packaging and assembly of virions.Assays for analyzing inhibition are described herein and are known inthe art.

An “RNA-dependent DNA polymerase” or “reverse transcriptase” is anenzyme that can synthesize a complementary DNA copy (“cDNA”) from an RNAtemplate. All known reverse transcriptases also have the ability to makea complementary DNA copy from a DNA template (target nucleic acid);thus, they are both RNA- and DNA-dependent DNA polymerases.

As used herein, a “label” or “detectable label” is any composition thatis detectable, either directly or indirectly, for example, byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. Useful labels include, but are not limited to, radioactiveisotopes (for example, 32p, 35S, and 3H), dyes, fluorescent dyes (forexample, Cy5 and Cy3), fluorophores (for example, fluorescein),electron-dense reagents, enzymes and their substrates (for example, ascommonly used in enzyme-linked immunoassays, such as, alkalinephosphatase and horse radish peroxidase), biotin-streptavidin,digoxigenin, or hapten; and proteins for which antisera or monoclonalantibodies are available. Moreover, a label or detectable moiety caninclude an “affinity tag” that, when coupled with the target nucleicacid and incubated with a test compound or compound library, allows forthe affinity capture of the target nucleic acid along with moleculesbound to the target nucleic acid. One skilled in the art will appreciatethat an affinity tag bound to the target nucleic acid has, bydefinition, a complimentary ligand coupled to a solid support thatallows for its capture. For example, useful affinity tags andcomplimentary partners include, but are not limited to,biotin-streptavidin, complimentary nucleic acid fragments (for example,oligo dT-oligo dA, oligo T-oligo A, oligo dG-oligo dC, oligo G-oligo C),aptamers, or haptens and proteins for which antisera or monoclonalantibodies are available. The label or detectable moiety is typicallybound, either covalently, through a linker or chemical bound, or throughionic, van der Waals or hydrogen bonds to the molecule to be detected.

The terms “alkyl”, “aryl” and other groups refer generally to bothunsubstituted and substituted groups unless specified to the contrary.

Unless specified otherwise, alkyl groups are hydrocarbon groups and arepreferably C₁-C₁₅ (that is, having 1 to 15 carbon atoms) alkyl groups,which can be branched or unbranched, acyclic or cyclic. The abovedefinition of an alkyl group and other definitions would apply also whenthe group is a substituent on another group (for example, an alkyl groupas a substituent of an alkylamino group or a dialkylamino group).

The term “aryl” refers to any functional group or substituent derivedfrom a simple aromatic ring, such as phenyl, thiophenyl, indoyl, etc.

The term “alkenyl” refers to a straight or branched chain hydrocarbongroup with at least one double bond, preferably with 2-15 carbon atoms.

The term “alkynyl” refers to a straight or branched chain hydrocarbongroup with at least one triple bond, preferably with 2-15 carbon atoms.

The terms “alkylene,” “alkenylene” and “alkynyllene” refer to bivalentforms of alkyl, alkenyl, and alkynyl groups, respectively.

The terms “halogen” or “halo” refer to fluoro, chloro, bromo, or iodo.

Substituent groups building off of the hydrocarbon groups includealkoxy, aryloxy, acyloxy, haloalkyl, perfluoroalkyl, fluorine, chlorine,bromine, carbamoyloxy, hydroxyl, nitro, cyano, cyanoalkyl, azido,azidoalkyl, formyl, hydrazine, hydroxyalkyl, alkoxyalkyl, and the like.

I. Antiviral Compounds

The compounds generally have one of the following formulas:

wherein:

-   Single or double bond, with the proviso that no allenes are intended    to be within the scope of the invention.-   M and N=1, 2 or 3 atoms from C, N, O-   X and Y═NR¹, O or S-   R¹═H, alkyl, aryl, aralkyl, alkaryl, heterocyclyl, heteroaryl,    substituted analogs thereof, wherein the substituents are selected    from the list of substituents, Z, defined herein.

Substituents Z as defined herein include C₁₋₆ alkyl (includingcycloalkyl), alkenyl, heterocyclyl, aryl, heteroaryl, halo (e.g., F, Cl,Br, or I), —OR′, —NR′R″, —CF₃, —CN, —NO₂, —C₂R′, —SR′, —N₃, —C(═O)NR′R″,—NR′C(═O)R″, —C(═O)R′, —C(═O)OR′, —OC(═O)R′, —OC(═O)NR′R″, —NR′C(═O)OR″,—SO₂R′, —SO₂NR′R″, and —NR′SO₂R″, where R′ and R″ are individuallyhydrogen, C₁₋₆ alkyl, cycloalkyl, heterocyclyl, aryl, or arylalkyl (suchas benzyl).

More specifically, within these broad formulas are the followingrepresentative narrower formulas:Ar₁—(CH₂)₂—NR₁—Ar₂  Formula A

wherein Ar₁ and Ar₂ are, independently, six membered aryl rings, five orsix membered ring heteroaryl rings, or analogs thereof in which a fivemembered heteroaryl or six membered aryl or heteroaryl ring is fused tothe six membered aryl rings, five or six membered ring heteroaryl rings,

n is 0 or 1, and

R₁ is H or a moiety cleaved in vivo to form H,

and each of the aryl/heteroaryl rings can be substituted with one tothree substituents, Z.

Substituents Z as defined herein include C₁₋₆ alkyl (includingcycloalkyl), alkenyl, heterocyclyl, aryl, heteroaryl, halo (e.g., F, Cl,Br, or I), —OR′, —NR′R″, —CF₃, —CN, —NO₂, —C₂R′, —SR′, —N₃, —C(═O)NR′R″,—NR′C(═O)R″, —C(═O)R′, —C(═O)OR′, —OC(═O)R′, —OC(═O)NR′R″, —NR′C(═O)OR″,—SO₂R′, —SO₂NR′R″, and —NR′SO₂R″, where R′ and R″ are individuallyhydrogen, C₁₋₆ alkyl, cycloalkyl, heterocyclyl, aryl, or arylalkyl (suchas benzyl).

In one embodiment, where one or more aryl rings are present, at leastone of the rings is an aniline or substituted aniline (i.e., an arylring with an —NH₂, primary amine, or secondary amine substituent).

Representative compounds from the table provided above, in which n is 0,Ar₁ is phenyl, and Ar₂ is pyridinyl or pyrimidinyl, include Compounds66, 73, and 111.

Compounds 16, 20, and 27 are compounds in which n is 0, Ar₁ and Ar₂ arepyrimidinyl, and one of the pyrimidinyl rings is fused to an aryl ring.

Compound 62 is a compound in which n is 0, Ar₁ is phenyl, and Ar₂ ispyridinyl, and the pyridinyl ring is fused to an aryl ring.

Compounds 6, 11, 12, and 90 are compounds in which n is 0, Ar₁ isphenyl, and Ar₂ is oxathiazole.

Compound 29, 39, and 61 are compounds in which n is 0, and Ar₁ and Ar₂are phenyl.

Compounds 25, 33, 37, 42, 51, 52, 53, 69, 79, 80, 87, 99, 100, 107, 112are compounds in which n is 1, and Ar₁ and Ar₂ are phenyl.

Compound 58 is a compound in which n is 1, and Ar₁ and Ar₂ are bothphenyl-fused heteroaryl rings.

Compounds 81 and 91 are compounds in which n is 1, Ar₁ is phenyl, andAr₂ is a phenyl-fused heteroaryl rings.

Compounds 4, 5, 13, and 15 have a core structure where n is 0, Ar₁ is1H-pyridin-4-one, and Ar₂ is a pyrimidine ring fused to a benzene ring,as shown below:

Additional representative compounds include the following:

where Ar₁, Ar₂, and R₁ are as defined above, m is 0, 1, 2 or 3, and thearyl/heteroaryl rings can be substituted with from 1 to 3 substituents,Z, as described above, with the proviso that at least one m is 2.

Specific embodiments are those in which one of m is 1 and the other m is2, both of m are 2, one of m is 0 and the other m is 2, and one of m is1 and the other m is 3. Compound 21 is an example of a compound whereone of m is 0 and the other m is 2. Compounds 19 and 36 are examples ofcompounds where one m is 1 and the other m is 2. Compounds 50 and 98 areexamples of compounds where one m is 1 and the other m is 3. Compounds50 and 98 both also include a benzofuran ring, and a phenyl ringsubstituted with a dimethylamine group at a position para to the linkageto the remainder of the molecule.

The following compounds either fall within Formula B, or are closelyrelated to Formula B:

Compound 46

The following compounds fall within Formula B-3 below:Ar-Q  B-3

wherein Q is selected from the group consisting of —NH—C(NR₁)—NH₂,—O—C(NR₁)—NH₂, —S—C(NR₁)—NH₂, —NH—C(O)—NH₂, —O—C(O)—NH₂, —S—C(O)—NH₂,—NH—C(S)—NH₂, O—C(S)—NH₂, and —S—C(S)—NH₂. Representative compoundsfalling within the scope of formula B-3 include the following:

where Z, j, and R₁ are as defined above.

The modifications of Formula B include the change from an amine linkageto an imine linkage or a C(O)CH₂CH₂NHC(O) linkage, or the replacement ofthe —(CH₂)_(m)—Ar₂ moiety with —C(NH)NH₂, where Ar is quinolone orisoquinoline These modifications can be applied across the range ofFormula B.

wherein m is 0, 1, or 2, X is NR₁, O, or S, and halo is F, Cl, Br, I. Inone embodiment of Formula B, X is S and halo is Cl. Representativeazacyclic rings include morpholine, azacyclopentane, and piperidine.

Compounds 31, 44, and 88 are examples of compounds of Formula C.

where Z, j and R₁ are as defined above, with the proviso that two R₁groups can link together to form a 5-7 membered ring azacyclic moiety.

Compounds 9 and 28 are examples of compounds of Formula D.

A structural analog of Formula D is shown below:

wherein R₁, Z and j are as defined above.

Representative compounds of Formula D-A are shown below:

where Z and j are as defined above.

Compounds 45 and 49 are examples of compounds of Formula E.

wherein Ar₁, R₁, Z and j are as defined above, and Ar₁ can include fromone to three Z substituents.

Compounds 48, 56, 64, and 77 are representative compounds of Formula F.

wherein X, R₁, Z, j, and n are as defined above, and the ═X moiety canbe present or not present (i.e., n is 0 or 1).

Compounds 17, 60, 74, and 96 are representative compounds within thescope of Formula G.

Alternatively, the compounds of Formula H can have the formula shownbelow, where the cyclohexadienone double bond is optional (as indicatedby a dashed line), as follows:

wherein the dashed line indicates the presence of an optional doublebond.

The analogs can have substantially any organic substituent or functionalgroup substituted in place of one or more of the hydrogen atoms on thering skeleton, for example, a substituent J as defined herein.

In one embodiment, R4, R5, R6, R7, R15, R16, and R17 are, independently,the same or different, and are selected from hydrogen, alkyl,cycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclic, heteroaryl,alkenyl, alkynyl, halo (F, Cl, Br, I), OR′, N(R′)₂, SR′, OCOR′, NHCOR′,N(COR′)COR′, SCOR′, OCOOR′, and NHCOOR′, wherein each R′ isindependently H, a lower alkyl (C₁-C₆), lower haloalkyl (C₁-C₆), loweralkoxy (C₁-C₆), lower alkenyl (C₂-C₆), lower alkynyl (C₂-C₆), lowercycloalkyl (C₃-C₆) aryl, heteroaryl, alkylaryl, or arylalkyl, whereinthe groups can be substituted with one or more substituents as definedabove).

Alternatively, one or more of R4 and R5, R5 and R6, R6 and R7, R15 andR16, and R16 and R17 together form a five, six, or seven-member ring,which ring can include one or more heteroatoms, such as O, S, and N(wherein N can be substituted with H or R′).

The compounds of Formula H use the following numbering scheme for thevarious positions:

In one aspect of this embodiment, one or both of the nitrogens atpositions 13 and 19 as listed above can be replaced with a CR′ moiety.

Naphtho[2′,3′:4,5]imidazo[1,2-a]pyridine-6,11-dione is a representativecompound of Formula H.

A similar structure to Formula H is shown below:

wherein n is 0, 1, or 2 and Z and j are as defined above.

Representative compounds are shown below:

Additional anthraquinone-type compounds fall within the formula shownbelow:

wherein R¹⁸ is amine, including 4-8 membered ring azacycles (cyclicamines), and Z and j are as defined above. In one embodiment, the Zsubstituent on the ring with the R¹⁸ moiety is H, alkyl, halo, or amine.

Representative compounds include the following:

Still further anthraquinones have the following formula:

wherein Z, j, R¹ and R² are as defined above.

Representative compounds include the following:

The analogs can have substantially any organic or inorganic substituentor functional group substituted in place of one or more of the hydrogenatoms on the ring skeleton (i.e., at positions 2, 3, 6, 7, and 8), forexample, a substituent J as defined herein.

In one embodiment, these substituents are, independently, the same ordifferent, and are selected from hydrogen, alkyl, cycloalkyl, aryl,arylalkyl, alkylaryl, heterocyclic, heteroaryl, alkenyl, alkynyl, halo(F, Cl, Br, I), OR′, N(R′)₂, SR′, OCOR′, NHCOR′, N(COR′)COR′, SCOR′,OCOOR′, and NHCOOR′, wherein each R′ is independently H, a lower alkyl(C₁-C₆), lower haloalkyl (C₁-C₆), lower alkoxy (C₁-C₆), lower alkenyl(C₂-C₆), lower alkynyl (C₂-C₆), lower cycloalkyl (C₃-C₆) aryl,heteroaryl, alkylaryl, or arylalkyl, wherein the groups can besubstituted with one or more substituents as defined above).

R1, R2, R3, and R4, are independently, the same or different, and areselected from hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, alkylaryl,heterocyclic, heteroaryl, alkenyl, alkynyl, —COR′, and —COOR′, whereinR′ is, independently H, a lower alkyl (C₁-C₆), lower haloalkyl (C₁-C₆),lower alkoxy (C₁-C₆), lower alkenyl (C₂-C₆), lower alkynyl (C₂-C₆),lower cycloalkyl (C₃-C₆) aryl, heteroaryl, alkylaryl, or arylalkyl,wherein the groups can be substituted with one or more substituents asdefined above).

In one embodiment, one or both of R1 and R2, and R3 and R4, togetherwith the nitrogens to which they are attached, form a 5-7 membered ring,which can include one or more additional heteroatoms such as O, S, or N,wherein the N can be bonded to a substituent R′, as defined above).

4,5-Bis(dimethylamino)-1-naphthaldehyde (Compound 10) is arepresentative compound of Formula I.

wherein, for compounds of Formulas J, K, and L, R₁, Z and j are asdefined above. In a specific embodiment of Formulas J and K, both of R₁adjacent the ring nitrogen are methyl. In another embodiment, both ofthese R₁ moieties link together to form a five, six, or seven-memberedring, which can optionally include a heteroatom such as O, S, or N.Compounds 14, 18, 22, and 24 are specific examples of Formula J.Compounds 23, 34, 35, 68, 82, and 85 are specific examples of Formula K.Compounds 30, 67, 78, and 103 are specific examples of Formula L.

wherein X, Z, j, m and R₁ are as defined above.

In a specific embodiment, X in the heteroaryl ring is O and/or X in thebridge between the aryl and heteroaryl ring is O. In one embodiment, nis 0, and in another embodiment, n is 1. Compounds 84 and 101 arespecific examples of Formula M, where n is 1. Compounds 63, 89, and 113are specific examples of Formula M, where n is 0. Examples ofheterocyclic rings that can be attached to the heteroaryl ring includepiperidine, piperazine, azacycloheptane, azacyclohexane,azacyclopentane, and morpholine.

wherein X, Z, j, are as defined above.

A specific subset of the compounds of Formula N have the followingformula:

That is, these compounds fall within the definition of Formula N, whereR₁ is alkaryl or aryl. In a specific embodiment, the X variables areselected so as to form a thiourea moiety. In another specificembodiment, at least one Z substituent is present on at least one arylring, and the substituent is an —NH₂, primary or secondary amine group.Compounds 76, 94, and 110 are specific examples of Formula M where n is0. Compounds 41 and 55 are specific examples of Formula N where n is 1.

Other compounds falling generally into the definition of Formula N,where R₁ is alkyl (in one case, cyclohexyl, a cycloalkyl moiety),include compounds 71 and 109.

wherein X, Z, j, and m are as defined above. In a specific embodiment,at least one aryl ring includes a nitro group. Compounds 54 and 93 arespecific examples of Formula M.

wherein Z, j, n, and R₁ are as defined above, and a) K is NR₁, or b) Kis N(R₁)₂, and the link to the other ring nitrogen is absent, in whichcase the other NR₁ moiety is an N(R₁)₂ moiety rather than an NR₁ moiety.

Specific compounds within the scope of Formula P include compounds 1 and10.

wherein Z, j, n, and X are as defined above, and R₂ is absent (i.e., adirect link between the aryl ring and the C═X moiety), or is an alkyl orcycloalkyl moiety linking the aryl ring and the C═X moiety. Compounds97, 104, 106, and 108 are examples of specific compounds falling withinFormula Q.

wherein X and R₁ are as defined elsewhere herein, o is an integer from 4to 8 (in compounds 2 and 3, the number is 5), R₂ is C₁₋₆ alkyl, and R₅is —C(═X)OR₁, —C(═X)SR₁, —C(═X)NHR₁, —X—C(═X)OR₁, —X—C(═X)SR₁,—X—C(═X)NHR₁, —O—R₁, —SR₁, or —NHR₁. In one embodiment, where the ringnitrogen is bound to R₁, the NR₁ is an amide moiety, and the amidemoiety functions as a prodrug form of compounds in which the NR1 is anamine.

wherein X is O or S, and Z, j, R₁, and Ar₁ are as defined above.

Representative compounds of Formula S are shown below:

wherein R₁, Z, and j are as defined above.

Representative compounds of Formula T are shown below:

wherein Z and R₁ are as defined above. In one embodiment, Z is C₁₋₆alkyl or O—C₁₋₆alkyl.

Representative compounds of Formula U and U-I are provided below:

wherein R₁₉ is NHC(O)Ar₁, and Z and j are as defined above.

Representative compounds of Formula V are shown below:

Compounds 8, 32, 40, 57, 75, 83, 95, and 114 do not fall within thescope of the various Formulas A through R. These compounds were alsoactive, and compounds of these formulas, where the aryl or heteroarylrings can be substituted with from one to three Z substituents are alsowithin the scope of the invention.

A number of compounds appear active in the assays described herein, andare all highly conjugated molecules. These compounds include Compounds7, 26, 38, 46, 47, 59, 65, 72, 86, 92, and 102. These compounds aregenerally types of compounds known to be highly conjugated, includingbenzophenones, triarylmethanes, fulvenes, and the like.

In each of these compounds, particularly those of Formula Q, an estergroup can be replaced with an amide or thioamide moiety to increase thein vivo stability. Carbamate, thiocarbamate, urea, thiourea, ether, andthioether moieties can also be substituted for ester moieties. Arylrings can be replaced with heteroaryl rings, such as thiophene rings inany of these compounds. For example, Compound 143 is an example of acompound that would fall within Formula Q if one of the aryl rings isreplaced with a thiophene ring, n is 0, and R₂ is a direct link betweenthe aryl ring and the C═X moiety. All of the compounds can optionally besubstituted with a morpholinyl or piperidinyl moiety, which can bedesirable to increase hydrophilicity.

In one embodiment, compounds of Formulas H and I are intended to bespecifically excluded.

Novel compounds may also be formed in a combination of substituentswhich creates a chiral center or another form of an isomeric center. Inthis embodiment, the compound may exist as a racemic mixture, a pureenantiomer, and any enantiomerically enriched mixture.

The compounds can occur in varying degrees of enantiomeric excess, andracemic mixtures can be purified using known chiral separationtechniques.

The compounds can be in a free base form or in a salt form (e.g., aspharmaceutically acceptable salts). Examples of suitablepharmaceutically acceptable salts include inorganic acid addition saltssuch as sulfate, phosphate, and nitrate; organic acid addition saltssuch as acetate, dichloroacetate, galactarate, propionate, succinate,lactate, glycolate, malate, tartrate, citrate, maleate, fumarate,methanesulfonate, p-toluenesulfonate, and ascorbate; salts with anacidic amino acid such as aspartate and glutamate; alkali metal saltssuch as sodium and potassium; alkaline earth metal salts such asmagnesium and calcium; ammonium salt; organic basic salts such astrimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine,and N,N′-dibenzylethylenediamine; and salts with a basic amino acid suchas lysine and arginine. The salts can be in some cases hydrates orethanol solvates. The stoichiometry of the salt will vary with thenature of the components.

Representative compounds include the following:3-ethyl-6-methoxy-1H-benzo[de]cinnoline, methyl6-(5-methyl-2-oxo-2,3-dihydro-1H-imidazol-4-yl)-6-oxohexanoate, ethyl6-(1-benzoyl-5-methyl-2-oxo-2,3-dihydro-1H-imidazol-4-yl)-6-oxohexanoate,2-[(8-ethoxy-4-methyl-2-quinazolinyl)amino]-5,6,7,8-tetrahydro-4(1H)-quinazolinone,2-[(6-methoxy-4-methyl-2-quinazolinyl)amino]-5,6-dimethyl-4(1H)-pyrimidinone,2-[(4,7-dimethyl-2-quinazolinyl)amino]-6-propyl-4(3H)-pyrimidinone,tris[4-(dimethylamino)phenyl]methanol,5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoic acid,N-ethyl-5-nitro-N-phenyl-2,1,3-benzoxadiazol-4-amine,4,5-bis(dimethylamino)-1-naphthaldehyde,N,N-dimethyl-N′-[4-(2-pyridinyl)-1,3-thiazol-2-yl]1,4-benzenediaminehydrobromide, N˜2˜-(4,6-dimethyl-2-pyrimidinyl)-2,4-quinazolinediaminehydrochloride,2-[(4,6,7-trimethyl-2-quinazolinyl)amino]-5,6,7,8-tetrahydro-4(1H)-quinazolinone,9-(2-methoxyphenyl)-2,3,7,7-tetramethyl-10-thioxo-9,10-dihydro-7H-isothiazolo[5,4-c]pyrrolo[3,2,1-ij]quinoline-4,5-dione,2-[(4,8-dimethyl-2-quinazolinyl)amino]-5,6,7,8-tetrahydro-4(1H)-quinazolinone,2-[(4-methyl-2-quinazolinyl)amino]-6-propyl-4(1H)-pyrimidinone,2-[(4-acetylphenyl)amino]-3-(1-pyrrolidinyl)naphthoquinone,9-(2,5-dimethoxyphenyl)-2-methoxy-7,7-dimethyl-10-thioxo-9,10-dihydro-7H-isothiazolo[5,4-c]pyrrolo[3,2,1-ij]quinoline-4,5-dione,[3-(1,3-benzodioxol-5-yl)-3-phenylpropyl][4-(dimethylamino)benzyl]aminehydrochloride,2-[(4,7-dimethyl-2-quinazolinyl)amino]-5-ethyl-6-methyl-4(3H)-pyrimidinone,N-[2-(1-cyclohexen-1-yl)ethyl]-2-(3-methylphenyl)-5-nitro-2H-1,2,3-triazol-4-amine3-oxide,2-methoxy-7,7-dimethyl-9-(2-propoxyphenyl)-10-thioxo-9,10-dihydro-7H-isothiazolo[5,4-c]pyrrolo[3,2,1-ij]quinoline-4,5-dione,4,4,6-trimethyl-1,2-dioxo-1,2-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-8-yl2-methylbenzoate,9-(2,3-dimethylphenyl)-2-methoxy-7,7-dimethyl-10-thioxo-9,10-dihydro-7H-isothiazolo[5,4-c]pyrrolo[3,2,1-ij]quinoline-4,5-dione,N′-(2-methoxybenzyl)-N,N-dimethyl-1,4-benzenediamine,2-(4-ethylphenyl)naphthoquinone,2-[(4,6-dimethyl-2-quinazolinyl)amino]-1,5,6,7-tetrahydro-4H-cyclopenta[d]pyrimidin-4-one,5-nitro-4-(1-piperidinyl)-2,1,3-benzoxadiazole,N,N-dimethyl-N′-[2-nitro-4-(trifluoromethyl)phenyl]-1,3-benzenediamine,1-[2-(benzyloxy)benzyl]-5-methyl-1H-indole-2,3-dione,1-[2-chloro-4-nitro-5-(vinylthio)-3-thienyl]pyrrolidine,2-(4-ethylphenyl)-5-methyl-4-[4-(4-morpholinyl)benzylidene]-2,4-dihydro-3H-pyrazol-3-one,(2-methoxybenzyl)[4-(1-piperidinyl)phenyl]amine,4,4,6-trimethyl-1,2-dioxo-1,2-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-8-yl2-methoxybenzoate,4,4,6-trimethyl-1,2-dioxo-1,2-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-8-yl2-chlorobenzoate,4-({[3-(2-furyl)-3-(2-methoxyphenyl)propyl]amino}methyl)-N,N-dimethylaniline,N′-[4-(allyloxy)-3-chloro-5-methoxybenzyl]-N,N-dimethyl-1,4-benzenediamine,2,6,7-trihydroxy-9-(5-nitro-2-furyl)-3H-xanthen-3-one,2-[ethyl(4-{[2-nitro-4-(trifluoromethyl)phenyl]amino}phenyl)amino]ethanol,5-[4-(dimethylamino)phenyl]-3-(4-methoxyphenyl)-N-methyl-4,5-dihydro-1H-pyrazole-1-carbothioamide,N-[4-(dimethylamino)phenyl]-N′-(4-methylbenzyl)thiourea,N-[4-(allyloxy)-3-methoxybenzyl]-4-(1-pyrrolidinyl)aniline,5-methyl-N-[7-(4-morpholinyl)-2,1,3-benzoxadiazol-4-yl]-4-phenyl-3-thiophenecarboxamide,4-[2-chloro-4-nitro-5-(vinylthio)-3-thienyl]morpholine,1-(4-fluorophenyl)-2-(2-nitrovinyl)-1H-pyrrole,1,1′-(2,4-cyclopentadien-1-ylidenemethylene)bis(4-methoxybenzene),3-(2-chlorophenyl)-6-ethyl-7-methoxy-4H-chromene-4-thione,N-[4-hydroxy-3-(phenylthio)-1-naphthyl]-4-methoxybenzenesulfonamide,1-(4-chlorophenyl)-2-(2-nitrovinyl)-1H-pyrrole,4-({[3-(2-furyl)-4-phenylbutyl]amino}methyl)-N,N-dimethylaniline,4-(4-benzyl-1-piperazinyl)-N-(4-fluorobenzyl)aniline;(2-ethoxy-3-methoxybenzyl)[4-(1-pyrrolidinyl)phenyl]amine,(4-fluorobenzyl)[4-(1-pyrrolidinyl)phenyl]amine,6-(dimethylamino)-2-(2-methylphenyl)-5-nitro-1H-benzo[de]isoquinoline-1,3(2H)-dione,N-(4-chlorobenzyl)-N′-[4-(diethylamino)phenyl]thiourea,4-fluoro-N-[4-hydroxy-3-(phenylthio)-1-naphthyl]benzenesulfonamide,2-bicyclo[2.2.1]hept-2-yl-5-nitro-1H-isoindole-1,3(2H)-dione,N-[2-(1H-indol-3-yl)ethyl]-9-acridinamine,6-bromo-2-(3-methoxy-4-propoxyphenyl)-3-nitro-2H-chromene, ethyl3-(4-methoxyphenyl)-1,4-dioxo-1,4-dihydro-2-naphthalenecarboxylate,(2-methoxyphenyl)[2-nitro-4-(trifluoromethyl)phenyl]amine,N′-(2,8-dimethyl-4-quinolinyl)-N,N-dimethyl-1,4-benzenediaminehydrochloride,2-(2-methylphenyl)-5-(1-piperidinyl)-1,3-oxazole-4-carbonitrile,N-[4-hydroxy-3-(phenylthio)-1-naphthyl]benzenesulfonamide,1-(4-chlorophenyl)-5-[(5-nitro-2-furyl)methylene]-2,4,6(1H,3H,5H)-pyrimidinetrione,N′-(4,6-dimethyl-2-pyrimidinyl)-N,N-dimethyl-1,4-benzenediamine,5-bromo-1-(2-chlorobenzyl)-1H-indole-2,3-dione,4,4,6-trimethyl-1,2-dioxo-1,2-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-8-yl2-thiophenecarboxylate,2-({[4-(dimethylamino)phenyl]amino}methyl)phenol,2-(2-fluorophenyl)-3-(1-methyl-1H-pyrrol-2-yl)acrylonitrile,N-[4-(diethylamino)phenyl]-N′-isobutylthiourea,2-(4-ethylphenyl)-4-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methyl-2,4-dihydro-3H-pyrazol-3-one,N-(2-methoxyphenyl)-3-nitro-2-pyridinamine, ethyl3-(4-methylphenyl)-1,4-dioxo-1,4-dihydro-2-naphthalenecarboxylate,5-{[(2-nitrophenyl)thio]amino}-1,3-benzodioxole,N-[4-(diethylamino)phenyl]-N′-(4-ethoxyphenyl)thiourea,N-[4-hydroxy-3-(phenylthio)-1-naphthyl]-2-thiophenesulfonamide,1-benzyl-5-bromo-7-methyl-1H-indole-2,3-dione,N-[4-(dimethylamino)benzyl]-6-methyl-2-pyridinamine,2-[2-bromo-4-({[4-(dimethylamino)phenyl]amino}methyl)-6-ethoxyphenoxy]-N-(tert-butyl)acetamide,N-[4-(dimethylamino)benzyl]-1-pentyl-1H-benzimidazol-2-amine,4,6-diethyl-4,8-dimethyl-4H-pyrrolo[3,2,1-ij]quinoline-1,2-dione,N-[(1-methyl-1H-pyrrol-2-yl)methylene]-4-(1-naphthylmethyl)-1-piperazinamine,5-(1-azepanyl)-2-[(3-chlorophenoxy)methyl]-1,3-oxazole-4-carbonitrile,6′-methyl-5′,6′-dihydrospiro[cyclohexane-1,4′-pyrrolo[3,2,1-ij]quinoline]-1′,2′-dione,4-(di-1H-indol-3-ylmethyl)-1,2-benzenediol,(5-bromo-2-methoxybenzyl)[4-(4-morpholinyl)phenyl]amine,1-[2-chloro-4-nitro-5-(vinylthio)-3-thienyl]piperidine,5-(1-azepanyl)-2-(2-fluorophenyl)-1,3-oxazole-4-carbonitrile,4-(2-{[4-(diethylamino)phenyl]amino}-1,3-thiazol-4-yl)-1,2-benzenediol,1-butyl-N-[4-(diethylamino)benzyl]-1H-benzimidazol-2-amine,(4-bromophenyl)[3-nitro-4-(1-piperidinyl)phenyl]methanone,2-(2,5-dimethylphenyl)-6-nitro-1H-benzo[de]isoquinoline-1,3(2H)-dione,N-[4-(diethylamino)phenyl]-N′-(2,4-dimethoxyphenyl)thiourea,5-(dimethylamino)-1,3-benzothiazole-2-thiol, ethyl3-(4-ethylbenzyl)-1,4-dioxo-1,4-dihydro-2-naphthalenecarboxylate,4-propylphenyl 4-nitrobenzoate,4-({[3-(2-furyl)-3-(4-methylphenyl)propyl]amino}methyl)-N,N-dimethylaniline,N-benzyl-5-(4-benzyl-1-piperazinyl)-2-nitroaniline,N-benzyl-4-chloro-2-nitroaniline,5-(1-azepanyl)-2-[(4-chlorophenoxy)methyl]-1,3-oxazole-4-carbonitrile,[4-(2,6-dimethyl-4-morpholinyl)-3-nitrophenyl](4-ethoxyphenyl)methanone,5-chloro-1-(2-chlorobenzyl)-1H-indole-2,3-dione, 2-fluorobenzyl2-chloro-4-nitrobenzoate,3-[(3,4-dimethylphenyl)amino]-1-(4-nitrophenyl)-1-propanone,N-[4-(diethylamino)phenyl]-2-phenylcyclopropanecarboxamide,2-[2-bromo-6-methoxy-4-({[4-(1-pyrrolidinyl)phenyl]amino}methyl)phenoxy]acetamide,N-[4-(1-azepanyl)phenyl]-2-methylbenzamide,N-cyclohexyl-N′-[4-(dimethylamino)phenyl]thiourea,N-(3-chloro-4-fluorophenyl)-N′-[4-(diethylamino)phenyl]thiourea,4,6-dimethyl-N-[4-(1-pyrrolidinyl)phenyl]-2-pyrimidinamine,(2,6-dichlorobenzyl)[4-(1-pyrrolidinyl)phenyl]amine,2-(2-fluorophenyl)-5-(4-phenyl-1-piperazinyl)-1,3-oxazole-4-carbonitrile,and1-[4-nitro-3-(1-pyrrolidinyl)phenyl]-4-(2-thienylcarbonyl)piperazine.

Specific compounds also includeN-{4-methyl-5-[(pentan-3-yl)carbamoyl]thiophen-2-yl}furan-2-carboxamide,2-[3-ethyl-4-oxo-5-(thiophen-3-ylmethylidene)-1,3-thiazolidin-2-ylidene]propanedinitrile,2-[(4-cyclopropyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)sulfanyl]-N,N-bis(propan-2-yl)propanamide,N-(cyclohexylmethyl)-3-[5,7-dimethyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl]propanamide,4-{4-[2,5-dimethyl-1-(1,2-oxazol-3-yl)-1H-pyrrol-3-yl]-1,3-thiazol-2-yl}-2,5,6-trimethyl-2,3-dihydropyridazin-3-one,2-methyl-N-(3-nitropyridin-2-yl)-1H-indol-5-amine,2-[(pyrimidin-2-ylsulfanyl)methyl]quinazolin-4-amine

Potential R-Group Substitutions:

Novel compounds may also be formed in the event that some combination ofsubstituents creates a chiral center or another form of an isomericcenter in any compound of the present list. The list would include anyor all of the racemic mixture, pure enantiomers, and anyenantiomerically enriched mixture.

For example, in one embodiment, the amino groups at the 4 and/or 5position on the naphthalene ring in the compounds of formula I can bereplaced with —C(R₁)₃, —OR₁, or —SR₁.

Representative compounds include the following:

-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(diethylamino)-1-naphthaldehyde-   4,5-bis(dipropylamino)-1-naphthaldehyde-   4,5-bis(dibutylamino)-1-naphthaldehyde-   4,5-bis(methylethylamino)-1-naphthaldehyde-   4,5-bis(methylpropylamino)-1-naphthaldehyde-   4,5-bis(methylbutylamino)-1-naphthaldehyde-   4,5-bis(ethylpropylamino)-1-naphthaldehyde-   4,5-bis(ethylbutylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4,5-bis(dimethylamino)-1-naphthaldehyde-   4-dimethylamino-5-diethylamino-1-naphthaldehyde-   4-dimethylamino-5-dipropylamino-1-naphthaldehyde-   4-dimethylamino-5-dibutylamino-1-naphthaldehyde-   4-diethylamino-5-dimethylamino-1-naphthaldehyde-   4-diethylamino-5-dipropylamino-1-naphthaldehyde-   4-diethylamino-5-dibutylamino-1-naphthaldehyde-   4-dipropylamino-5-dimethylamino-1-naphthaldehyde-   4-dipropylamino-5-diethylamino-1-naphthaldehyde-   4-dipropylamino-5-dibutylamino-1-naphthaldehyde.    II. Synthetic Methods

The compounds described herein all include at least one aryl orheteroaryl ring, and all of these rings can be further substituted withone or more substituents, as defined herein. Those skilled in the artwill readily understand that incorporation of other substituents onto anaryl or heteroaryl ring used as a starting material to prepare thecompounds described herein, and other positions in the compoundframework, can be readily realized. Such substituents can provide usefulproperties in and of themselves or serve as a handle for furthersynthetic elaboration.

Benzene rings (and pyridine, pyrimidine, pyrazine, and other heteroarylrings) can be substituted using known chemistry, including the reactionsdiscussed below. For example, the nitro group on nitrobenzene can bereacted with sodium nitrite to form the diazonium salt, and thediazonium salt manipulated as discussed above to form the varioussubstituents on a benzene ring.

Diazonium salts can be halogenated using various known procedures, whichvary depending on the particular halogen. Examples of suitable reagentsinclude bromine/water in concentrated HBr, thionyl chloride, pyr-ICl,fluorine and Amberlyst-A

A number of other analogs, bearing substituents in the diazotizedposition, can be synthesized from the corresponding amino compounds, viathe diazocyclopentadiene intermediate. The diazo compounds can beprepared using known chemistry, for example, as described above.

The nitro derivatives can be reduced to the amine compound by reactionwith a nitrite salt, typically in the presence of an acid. Othersubstituted analogs can be produced from diazonium salt intermediates,including, but are not limited to, hydroxy, alkoxy, fluoro, chloro,iodo, cyano, and mercapto, using general techniques known to those ofskill in the art.

For example, hydroxy-aromatic/heteroaromatic analogues can be preparedby reacting the diazonium salt intermediate with water. Halogens on anaryl or heteroaryl ring can be converted to Grignard or organolithiumreagents, which in turn can be reacted with suitable aldehyde or ketoneto form alcohol-containing side chains. Likewise, alkoxy analogues canbe made by reacting the diazo compounds with alcohols. The diazocompounds can also be used to synthesize cyano or halo compounds, aswill be known to those skilled in the art. Mercapto substitutions can beobtained using techniques described in Hoffman et al., J. Med. Chem. 36:953 (1993). The mercaptan so generated can, in turn, be converted to analkylthio substituent by reaction with sodium hydride and an appropriatealkyl bromide. Subsequent oxidation would then provide a sulfone.Acylamido analogs of the aforementioned compounds can be prepared byreacting the corresponding amino compounds with an appropriate acidanhydride or acid chloride using techniques known to those skilled inthe art of organic synthesis.

Hydroxy-substituted analogs can be used to prepare correspondingalkanoyloxy-substituted compounds by reaction with the appropriate acid,acid chloride, or acid anhydride. Likewise, the hydroxy compounds areprecursors of both the aryloxy and heteroaryloxy via nucleophilicaromatic substitution at electron deficient aromatic rings. Suchchemistry is well known to those skilled in the art of organicsynthesis. Ether derivatives can also be prepared from the hydroxycompounds by alkylation with alkyl halides and a suitable base or viaMitsunobu chemistry, in which a trialkyl- or triarylphosphine anddiethyl azodicarboxylate are typically used. See Hughes, Org. React.(N.Y.) 42: 335 (1992) and Hughes, Org. Prep. Proced. Int. 28: 127 (1996)for typical Mitsunobu conditions.

Cyano-substituted analogs can be hydrolyzed to afford the correspondingcarboxamido-substituted compounds. Further hydrolysis results information of the corresponding carboxylic acid-substituted analogs.Reduction of the cyano-substituted analogs with lithium aluminum hydrideyields the corresponding aminomethyl analogs. Acyl-substituted analogscan be prepared from corresponding carboxylic acid-substituted analogsby reaction with an appropriate alkyllithium using techniques known tothose skilled in the art of organic synthesis.

Carboxylic acid-substituted analogs can be converted to thecorresponding esters by reaction with an appropriate alcohol and acidcatalyst. Compounds with an ester group can be reduced with sodiumborohydride or lithium aluminum hydride to produce the correspondinghydroxymethyl-substituted analogs. These analogs in turn can beconverted to compounds bearing an ether moiety by reaction with sodiumhydride and an appropriate alkyl halide, using conventional techniques.Alternatively, the hydroxymethyl-substituted analogs can be reacted withtosyl chloride to provide the corresponding tosyloxymethyl analogs,which can be converted to the corresponding alkylaminoacyl analogs bysequential treatment with thionyl chloride and an appropriatealkylamine. Certain of these amides are known to readily undergonucleophilic acyl substitution to produce ketones.

Hydroxy-substituted analogs can be used to prepare N-alkyl- orN-arylcarbamoyloxy-substituted compounds by reaction with N-alkyl- orN-arylisocyanates. Amino-substituted analogs can be used to preparealkoxycarboxamido-substituted compounds and urea derivatives by reactionwith alkyl chloroformate esters and N-alkyl- or N-arylisocyanates,respectively, using techniques known to those skilled in the art oforganic synthesis.

Synthesis of Compounds of Formula A

The compounds of Formula A include an aryl or heteroaryl ring linked toanother aryl or heteroaryl ring to form an amine. Where R₁ is H, thecompounds are secondary amines. Where R₁ is other than H, the compoundsare tertiary amines. Where n is 0, the amine nitrogen is linked directlyto the Ar₁ ring, and where n is 1, a methylene bridge exists between theamine nitrogen and the Ar₁ ring (i.e., a benzylamine when Ar₁ is abenzene ring). The formation of aniline moieties is well known, asdiscussed above with respect to forming amine substituents onaryl/heteroaryl rings. The formation of a benzylamine can take place byreacting a benzyl halide with an amine using standard nucleophilicdisplacement chemistry.

Synthesis of Compounds of Formula B

The compounds of Formula B include an aryl or heteroaryl ring linked toanother aryl or heteroaryl ring to form an amine. Where R₁ is H, thecompounds are secondary amines. Where R₁ is other than H, the compoundsare tertiary amines. Where n is 0, the amine nitrogen is linked directlyto the Ar₁ ring, and where n is 1, 2, or 3, an alkylene bridge existsbetween the amine nitrogen and the Ar₁ ring (i.e., a benzylamine,benzethylamine, and the like, when Ar₁ is a benzene ring). The formationof aniline moieties is well known, as discussed above with respect toforming amine substituents on aryl/heteroaryl rings. The formation of abenzylamine, benzethylamine, and the like can take place by reacting aarylalkyl halide with an amine using standard nucleophilic displacementchemistry.

Synthesis of Compounds of Formula C

The compounds of Formula C are 5-membered ring heteroaryl compoundswhich include various substituents at various positions on the rings.The nitro group at position 3 can be difficult to attach to anunsubstituted 5-membered ring heteroaryl, since the preference fornitration can be at the 2-position. However, once desired halosubstituents are placed on 2 and 5 position on the ring, for example, byreacting the heteroaryl ring with an elemental halogen in the presenceof acetic acid, the ring can be nitrated at the 3-position. The halogenat the 5-position can be reacted with vinyl sulfide in a nucleophilicdisplacement reaction to form the S-vinyl ether. A halogenation reactionwill place a halogen at the 3-position, which can be displaced using asuitable amine to form the heterocyclic ring attached to the heteroarylring.

Synthesis of Compounds of Formula D

The compounds of Formula D can be formed by starting with theunsubstituted ring structure, and performing a nitration reaction. Then,halogenation can be used to place a halogen at a position adjacent tothe nitro group, and the halogen can be displaced with an amine to formthe compounds of Formula D.

Synthesis of Compounds of Formula E

The compounds of Formula E include a haloaryl ring with a pyrrole ringattached para to the halogen. The pyrrole ring includes a CH₂═CHNO₂moiety at the 2-position. This moiety can be provided, for example, bystarting with a pyrrole with a CHO group at the 2-position, and usingWittig chemistry to attach the CH₂═CHNO₂ moiety. An aryl (i.e., phenyl)ring with a halogen at the 1-position and a diazonium salt at the4-position can be reacted with the pyrrole to form a linkage between thepyrrole ring and the aryl ring.

Synthesis of Compounds of Formula F

The compounds of Formula F include a naphthyl ring that further includesan aryl sulfonamide moiety, an aryl thioether moiety, and an —OH orether moiety. Starting from a naphthyl ring with appropriatelypositioned amine, thiol, and hydroxy groups, one can selectively protecttwo of the three groups. A thiol group can be reacted with a diazoniumgroup on a benzene ring to form the aryl thioether. The amine group canbe reacted with an aryl sulfonyl halide to form the sulfonamide moiety.The protected hydroxy group can be deprotected to form an OH group,which can then be converted to ethers or esters if desired, using knownchemistry. Since an amine group is more nucleophilic than a hydroxygroup, the sulfonamide can likely be prepared even in the presence of anunprotected hydroxy group.

Synthesis of Compounds of Formula G

The compounds of Formula G are napthoquinones. They can typically beprepared from appropriately substituted 1,4-quinones and dienes usingDiels Alder chemistry (see, for example, Witayakron et al., TetrahedronLetters, Volume 48, Issue 17, 23 Apr. 2007, Pages 2983-2987, thecontents of which are hereby incorporated by reference).

Synthesis of Compounds of Formula H

The compounds of Formula H are also substituted napthoquinones. They cansimilarly be prepared from suitably appropriately substituted1,4-quinones and dienes using Diels Alder chemistry. The quinones areprepared from 1,4-bisphenols, and the additional ring functionality canbe incorporated by starting with 1,4-bisphenols with an amine group atthe 2 position, and a pyridine carboxamide at the 3-position, where animine linkage is formed between the carboxy group on the carboxamide andthe amine at the 2-position.

Synthesis of Compounds of Formula I

The compounds of Formula I are naphthalenes with amines at the 4 and 5position, and an aldehydes at the 1-position. Ideally, the amines aredialkylamines, so that they do not react with the aldehyde moiety toform an intramolecular imine group. Amine groups are typically formed onaromatic rings by a combination of nitration with nitric acid, andreduction of the nitro group to an amine group. Alkylation of the aminegroups involves routine nucleophilic displacement chemistry withappropriate alkylamines, whereas arylation can involve reaction of anamine with a diazonium salt. The aldehydes moiety can be introduced byreacting an organolithium reagent (a naphthyl-lithium) with isonitrilesto the corresponding lithium aldimine. Subsequent hydrolysis effectivelyconverts the organolithium compound to its aldehydes (see, for example,G. E. Niznik, W. H. Morrison, III, and H. M. Walborsky (1988),“1-d-Aldehydes from Organometallic Reagents: 2-Methylbutanal-1-d”, Org.Synth., Coll. Vol. 6: 751, the contents of which are hereby incorporatedby reference).

Synthesis of Compounds of Formula J

The compounds of Formula J can be formed from appropriatelyfunctionalized dihydroquinolines. The amine in the dihydroquinoline canbe reacted with oxaloyl chloride in a stepwise fashion, and theremaining acid chloride can be reacted with the aromatic ring viaFriedel-Crafts acylation conditions to form the cyclic structure. Fromthere, routine chemistry, for example, 3,2 Diels Alder chemistryheterocyclic ring structure, for example, by stepwise reaction of ananiline with appropriately functionalized groups on the dihydroquinolineframework.

Synthesis of Compounds of Formula K

The compounds of Formula K can be formed from appropriatelyfunctionalized dihydroquinolines. The amine in the dihydroquinoline canbe reacted with oxaloyl chloride in a stepwise fashion, and theremaining acid chloride can be reacted with the aromatic ring viaFriedel-Crafts acylation conditions to form the cyclic structure.

Synthesis of Compounds of Formula L

The compounds of Formula L can be formed from appropriatelyfunctionalized anilines. The amine in the aniline can be reacted withoxaloyl chloride in a stepwise fashion, and the remaining acid chloridecan be reacted with the aromatic ring via Friedel-Crafts acylationconditions to form the cyclic structure. From there, the amine canfurther react with an appropriately functionalized benzyl halide to formthe benzylamine moiety.

Synthesis of Compounds of Formula M

The compounds of Formula M can be formed from appropriatelyfunctionalized 5-membered ring heteroaryls. The amine moiety can beformed by initial nitration, which tends to form nitro groups in the2-position, and subsequent reduction to an amine group (which may bepostponed until the other moieties are present). Halogenation occurringafter the nitration step can place a halo group at the 3-position, whichcan then be nucleophilically displaced by a cyanide ion to form thenitrile, or converted to an organolithium reagent and reacted, forexample, with cyanogen bromide to form the nitrile moiety. The sidechain (alkylaryl, ether, and the like) can be incorporated usingstandard chemistry, such as nucleophilic substitution using anorganolithium reagent.

Synthesis of Compounds of Formula N

The compounds of Formula N include urea, thiourea, and other similarmoieties. At least one of these moieties includes an O, S, or N linkedto an aryl ring, so the compounds can be synthesized from anappropriately functionalized phenyl isocyanate, thioisocyanate, and thelike by nucleophilic reaction with an appropriately functionalizedamine, thiol, or hydroxy-containing material (i.e., R₁—XH).

Synthesis of Compounds of Formula O

The compounds of Formula O include a naphthalene ring, and a cyclic ringstructure including an imide moiety. The compounds can be prepared fromnaphthalene dicarboxylic acids and a suitably functionalized aniline inmuch the same way as phthalimide is formed (i.e., ring cyclization asthe amine reacts with the acids, or activated forms thereof).Alternatively, the acids or activated forms thereof, such as anhydrides,acid chlorides, and the like, can be reacted with ammonia, which is thenreacted with an aryl-diazonium salt to form the aniline.

Synthesis of Compounds of Formula P

The compounds of Formula P include a naphthalene ring, and a) a cyclicring structure including two ring nitrogens originating at positions 1and 8 on the naphthalene ring, b) a cyclic ring structure including onering nitrogen originating at position 1 or 8 on the naphthalene ring,and a methylamine moiety at the other of these positions, or c) twoamines, at positions 1 and 8 on the naphthalene ring. Naphthalene 1,8diamine is a commercially available compound whose synthesis need not bediscussed herein. Alkylamines can be formed by reacting an amine (orammonia) with a —CH₂Br moiety at the 1 or 8 position, or with anothernaphthyl halide at this position. Rings with adjacent ring nitrogens canbe formed, for example, by step-wise reaction of suitably functionalizedhydrazines with diazonium salts (to form a linkage directly on anaromatic ring) or a —CH₂Br moiety on the naphthalene ring (or othersuitable leaving group other than bromide on such moiety).

Synthesis of Compounds of Formula Q

The compounds of Formula Q include an amide, ester, thioester, orsimilar linkage, where to the left and right of thecarbonyl/thiocarbonyl moiety lie an aryl or arylalkyl moiety. Thesecompounds can be prepared from suitably functionalized benzoic acid orphenyl-alkanoic acid by forming an acid halide or anhydride (or versionsthereof where the carbonyl is replaced by a C(═S) or C(═NR₁) moiety),and reacting with a suitably functionalized phenol, thiophenol, aniline,or aryl-substituted hydroxyalkane, thioalkane, or amine.

Synthesis of Compounds of Formula R

The compounds of Formula R are functionalized cyclic ureas. They can beformed from suitably functionalized diamines (with amine moieties onadjacent carbon atoms) by reaction with phosgene, diphosgene,triphosgene, and the like. The carbonyl side chain can be formed, forexample, by converting a carboxylic acid to an acid halide, and reactingthe acid halide with a suitable Grignard or organolithium reagent.

Synthesis of Compounds of Formula S

The compounds of Formula S are functionalized cyclic ureas. They can beformed from suitably functionalized rings of the formula:

by deprotonation with a strong base, followed by reaction with acompound of the formula:

The resulting alcohol can then be dehydrated to form the compounds ofFormula S.

Synthesis of Compounds of Formula T

The compounds of Formula T can be formed by reacting a compound of theformula:

with a suitable hydrazine of the formula:

Additional synthetic details are provided below:

Synthesis of Compounds of Formula V

The compounds of Formula V can be prepared by reacting a compound offormula:

with a suitable strong base, and reacting the resulting carbanion with asuitably functionalized benzaldehyde. The resulting benzyl alcohol canbe dehydrated to form the compounds of Formula V.

Preparing the 4,5-Bis(Diamino)-1-Naphthaldehyde Framework

1,8-diamino naphthalene is commercially available, and is used as astarting material for other commercially available analogs, such asproton sponge (1,8-bis-dimethylamino naphthalene).

To prepare the simplest analog, where R1-R4 are H, one can react1,8-diamino naphthalene with carbon monoxide in a Friedel Craft reactioncan produce the formyl group at a para-position to one of the aminogroups. See, for example, “Aldehyde Syntheses” G. A. Olah, et al.,Friedel-Crafts and Related Reactions, Wiley-Interscience, vol. III,Chapter XXXVIII, pp. 1153-1256, 1964. “Superacid-Catalyzed Formylationof Aromatics with Carbon Monoxide,” G. A. Olah et al., J. Org. Chem.,vol. 50, pp. 1483-1486, 1985. Note that the numbering on the naphthalenering changes as the formyl substituent is added (i.e., formyl becomesthe 1-position, and the amino groups are numbered accordingly, goingfrom 1,8-diamino to 4,5-diamino). This chemistry is shown below inScheme 1.

N-Alkylation/Arylation

Either before or after the Friedel Crafts reaction to put the formylsubstituent on the naphthalene ring, one can react one or both of theamino groups (—NH₂) with an alkylating reagent to alkylate one or bothof the amine groups, depending on stoichiometry.

For example, proton sponge (the bis-dimethylamino analogue of1,8-diaminonaphthalene) is prepared by reacting 1,8-diaminonaphthalenewith dimethyl sulfate.

Suitably functionalized aryl groups (i.e., aryl rings with any desiredsubstitution) can be prepared that include a diazonium moiety at theposition in which it is desired to attach the aryl group to the aminemoiety(ies) on the 1,8-diaminonaphthalene. The amine moiety(ies) canthen displaced the diazonium moiety to provide aryl amines.

Protecting groups can be used when it is desirable to alkylate/arylateone amino group in preference to the other. For example, one canselectively protect either the 1-amine or the 8-amine in the1,8-diaminonaphthalene starting material, for example, using a t-boc orother protecting groups, such as those described in Greene and Wuts,Protective Groups in Organic Synthesis, 3rd Edition, June 1999, JohnWiley & Sons Inc., the contents of which are hereby incorporated byreference. Then, following the alkylation/arylation reaction(s), theprotective groups can be removed. The aldehyde group can be protected,for example, as an acetal group, which can be deprotected at a latertime by simply reacting the acetal with water in the presence of an acidcatalyst.

Substitution at Positions 2, 3, 6, 7, and 8

Where it is desirable to provide substitution at positions 2, 3, 6, 7,and 8 on the naphthalene ring, electrophilic aromatic substitution canbe used to provide other desired functionality. For example, alkyl,aryl, heteroaryl, alkaryl, arylalkyl, alkenyl, alkynyl, and acyl groupscan be added using Friedel-Crafts alkylation/arylation/acylationreactions. Other electrophilic aromatic substitution reactions can beused, for example, to provide halogens, such as by forming chloronium orbromonium ions in situ and reacting them with the aromatic ring, or byforming sulfonium or nitronium ions to provide sulfonyl or nitro groups.

Friedel Crafts alkylation is conducted using an appropriate halo-alkylmoiety, and a Lewis acid. The alkyl moiety forms a carbocation, andelectrons from the aryl ring form a bond with the carbocation, placing apositive charge on the aryl ring. The aryl ring then loses a proton.Alkyl and alkaryl moieties (such as benzyl moieties) can be added inthis fashion.

Friedel Crafts acylation is similar, but uses an acid halide, such as anacid chloride, to place a ketone moiety on the ring. The acid halide canbe an alkyl acid, such as acetic acid, propionic acid, butyric acid, andthe like, or can be an aromatic acid, such as benzoic acid, p-toluicacid, and the like.

Friedel Crafts arylation (also known as the Scholl reaction) is acoupling reaction with two aryl rings, catalyzed by a Lewis acid. Theproton lost during the coupling reaction serves as an additionalcatalyst. Typical Reagents are iron(III) chloride in dichloromethane,copper(II) chloride, PIFA and boron trifluoride etherate indichloromethane, Molybdenum(V) chloride and lead tetraacetate with BF₃in acetonitrile.

Substitution typically occurs at a position ortho or para to the aminegroups. So, positions 3, 6, and 8 are typically functionalized usingthis chemistry. Substitution of the naphthalene ring at a meta positionto the amine groups (i.e., positions 2 and 7) can be performed byoxidizing the amine group(s) to nitro groups, which leads to metasubstitution. The nitro groups can then be reduced back to the aminegroups.

Formation of Heterocyclic Rings Incorporating the Amino Groups

Either or both of the amino groups in the 1,8-diamino naphthalenestarting material, or in 4,5-bis(amino)-1-naphthaldehyde, can becyclized using a di-halo compound. For example, a five membered ring canbe formed using nucleophilic substitution. The amine is reacted with a1,4-di-halobutane, such as 1,4-dibromobutane, and a six membered ringcan be formed using a 1,5-dihalopentane, such as 1,5-dibromopentane. Thereaction typically takes place in the presence of a tertiary amine,which reacts with the in situ-formed hydrogen halide, such as hydrogenbromide.

When it is desirable to incorporate an additional heteroatom into thecyclic group, one or more of the carbons in the dihaloalkane can bereplaced with a heteroatom, such as O, S, or N (where the N can besubstituted with an alkyl, aryl, alkaryl, aralkyl, or other suchsubstituent).

Preparation of the Naphtho[2′,3′:4,5]imidazo[1,2-a]pyridine-6,11-dioneFramework

The compound 2,3-dichloro-1,4-naphthoquinone is commercially available,and is described, for example, in Honda, Nakanishi and Tabe, Bull. ChemSoc. Japan, 56(8):2338-2340 (1983), the contents of which are herebyincorporated by reference.

The carbonyl moieties on this compound can be protected, for example, asan ethylene ketal, and then reacted with 2-hydroxypyridine, the tautomerof which has the formula:

to provide an intermediate in which the pyridine nitrogen reacts withone of the ring halogens. Nucleophilic displacement of the halogen withammonia (or an amine, if an N-alkyl, N-aryl, or other such derivative isdesired) affords an amine group, which cyclizes with the carbonyl moietyin the pyridine to form an imine linkage in a ring-closure step.Deprotection of the ketone moieties, and catalytic dehydrogenation,afford the final product. This chemistry is shown below in Scheme 2.

As described above with respect to formation of the4,5-bis(diamino)-1-naphthaldehyde compounds, substitution on thearomatic ring (substituents R4-R7) can be performed using Friedel Craftsalkylation, acylation, or arylation, or other known electrophilicaromatic substitution. Substitution of the pyridine ring (i.e., R15-17)can be performed using well-known substitution reactions for producingpyridine analogs. These substitution reactions include electrophilicaromatic substitution, and nucleophilic aromatic substitution reactions.

Electrophilic Aromatic Substitution on Pyridine

Electrophiles react preferentially with the lone pair of the nitrogen togenerate the pyridinium ion which, being positively charged, isunreactive towards electrophilic substitution. Neutral pyridine, whichcan react with electrophiles, is present only in a very low equilibriumconcentration, so the rate of electrophilic aromatic substitutionreactions is slow relative to aromatic rings.

The ring nitrogen polarizes the p-electron system, resulting indecreased electron density on the carbons, and as a result,electrophilic substitution typically forms 3-substituted products (the3-position is the least disfavored position). This is analogous to how anitro-substituent directs electrophilic substitution of benzene to themeta position.

Nucleophilic Aromatic Substitution on Pyridine

Pyridines are susceptible to nucleophilic attack at C-2 and C-4. Byanalogy with nitrobenzene, 2- or 4-halopyridines will undergopreferential substitution of the halide, compared to 3-halopyridines.Strongly basic nucleophiles, such as NH₂ ⁻, and alkyllithium andaryllithium or comparable Grignard reagents, will add at C-2 to form the2-substituted pyridine, even without a leaving group. Where thenucleophile is NH₂ ⁻, the reaction is known as the Chichibabin reaction.

Enantiomeric Purification

As used herein, the term “enantiomerically pure” refers to a nucleotidecomposition that comprises at least approximately 95%, and, preferably,approximately 97%, 98%, 99% or 100% of a single enantiomer of thatnucleotide.

As used herein, the term “substantially free of” or “substantially inthe absence of” refers to a nucleotide composition that includes atleast 85 to 90% by weight, preferably 95% to 98% by weight, and, evenmore preferably, 99% to 100% by weight, of the designated enantiomer ofthat nucleotide. In a preferred embodiment, the compounds describedherein are substantially free of enantiomers.

Similarly, the term “isolated” refers to a nucleotide composition thatincludes at least 85 to 90% by weight, preferably 95% to 98% by weight,and, even more preferably, 99% to 100% by weight, of the nucleotide, theremainder comprising other chemical species or enantiomers.

The compounds described herein may have asymmetric centers and occur asracemates, racemic mixtures, individual diastereomers or enantiomers,with all isomeric forms being included in the present invention.Compounds of the present invention having a chiral center can exist inand be isolated in optically active and racemic forms. Some compoundscan exhibit polymorphism. The present invention encompasses racemic,optically-active, polymorphic, or stereoisomeric forms, or mixturesthereof, of a compound of the invention, which possess the usefulproperties described herein. The optically active forms can be preparedby, for example, resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase or by enzymatic resolution. One can either purify therespective nucleoside, then derivatize the nucleoside to form thecompounds described herein, or purify the nucleotides themselves.

Optically active forms of the compounds can be prepared using any methodknown in the art, including but not limited to by resolution of theracemic form by recrystallization techniques, by synthesis fromoptically-active starting materials, by chiral synthesis, or bychromatographic separation using a chiral stationary phase.

Examples of methods to obtain optically active materials include atleast the following.

-   -   i) physical separation of crystals: a technique whereby        macroscopic crystals of the individual enantiomers are manually        separated. This technique can be used if crystals of the        separate enantiomers exist, i.e., the material is a        conglomerate, and the crystals are visually distinct;    -   ii) simultaneous crystallization: a technique whereby the        individual enantiomers are separately crystallized from a        solution of the racemate, possible only if the latter is a        conglomerate in the solid state;    -   iii) enzymatic resolutions: a technique whereby partial or        complete separation of a racemate by virtue of differing rates        of reaction for the enantiomers with an enzyme;    -   iv) enzymatic asymmetric synthesis: a synthetic technique        whereby at least one step of the synthesis uses an enzymatic        reaction to obtain an enantiomerically pure or enriched        synthetic precursor of the desired enantiomer;    -   v) chemical asymmetric synthesis: a synthetic technique whereby        the desired enantiomer is synthesized from an achiral precursor        under conditions that produce asymmetry (i.e., chirality) in the        product, which can be achieved using chiral catalysts or chiral        auxiliaries;    -   vi) diastereomer separations: a technique whereby a racemic        compound is reacted with an enantiomerically pure reagent (the        chiral auxiliary) that converts the individual enantiomers to        diastereomers. The resulting diastereomers are then separated by        chromatography or crystallization by virtue of their now more        distinct structural differences and the chiral auxiliary later        removed to obtain the desired enantiomer;    -   vii) first- and second-order asymmetric transformations: a        technique whereby diastereomers from the racemate equilibrate to        yield a preponderance in solution of the diastereomer from the        desired enantiomer or where preferential crystallization of the        diastereomer from the desired enantiomer perturbs the        equilibrium such that eventually in principle all the material        is converted to the crystalline diastereomer from the desired        enantiomer. The desired enantiomer is then released from the        diastereomer;    -   viii) kinetic resolutions: this technique refers to the        achievement of partial or complete resolution of a racemate (or        of a further resolution of a partially resolved compound) by        virtue of unequal reaction rates of the enantiomers with a        chiral, non-racemic reagent or catalyst under kinetic        conditions;    -   ix) enantiospecific synthesis from non-racemic precursors: a        synthetic technique whereby the desired enantiomer is obtained        from non-chiral starting materials and where the stereochemical        integrity is not or is only minimally compromised over the        course of the synthesis;    -   x) chiral liquid chromatography: a technique whereby the        enantiomers of a racemate are separated in a liquid mobile phase        by virtue of their differing interactions with a stationary        phase (including but not limited to via chiral HPLC). The        stationary phase can be made of chiral material or the mobile        phase can contain an additional chiral material to provoke the        differing interactions;    -   xi) chiral gas chromatography: a technique whereby the racemate        is volatilized and enantiomers are separated by virtue of their        differing interactions in the gaseous mobile phase with a column        containing a fixed non-racemic chiral adsorbent phase;    -   xii) extraction with chiral solvents: a technique whereby the        enantiomers are separated by virtue of preferential dissolution        of one enantiomer into a particular chiral solvent;    -   xiii) transport across chiral membranes: a technique whereby a        racemate is placed in contact with a thin membrane barrier. The        barrier typically separates two miscible fluids, one containing        the racemate, and a driving force such as concentration or        pressure differential causes preferential transport across the        membrane barrier. Separation occurs as a result of the        non-racemic chiral nature of the membrane that allows only one        enantiomer of the racemate to pass through.

Chiral chromatography, including but not limited to simulated moving bedchromatography, is used in one embodiment. A wide variety of chiralstationary phases are commercially available.

III. Methods of Treatment

The compounds described herein are capable of inhibiting viralpropagation. The retroviral propagation can be inhibited by inhibitingretroviral reverse transcription, viral recruitment of the retroviralprimer used in translation, human tRNA^(Lys3), inhibiting the finalpackaging and assembly of new virions, and/or inhibiting the binding ofa host cell tRNA to a target nucleic acid molecule.

Accordingly, these compounds can be used in methods to treat patientssuffering from retroviral infections. That is, a retroviral viralinfection can be treated or prevented by administering one or moreinhibitors of retroviral propagation, for example, inhibitors ofretroviral reverse transcription, binding to host cell tRNA and a targetnucleic acid molecule, recruitment of the retroviral primer, humantRNA^(Lys3), viral RNA translation into viral proteins, and final viralpackaging and assembly of virions. Treatment of viral disease has notbeen heretofore accomplished by using such inhibitors.

The compounds can be used to treat or prevent viral infections,including infections by retroviruses, and/or to inhibit viralreplication, propagation, reverse transcription, mRNA translation,and/or final viral packaging and assembly. Retroviruses for whichinhibitors can be identified by the methods disclosed herein include anyviruses having RNA as their primary genetic material and use reversetranscription to produce DNA. Such viruses include, but are not limitedto, Feline Immunodeficiency Virus (FIV), Simian Immunodeficiency Virus(SIV), Avian Leucosis Virus, Feline Leukemia Virus, Walleye DermalSarcoma Virus, Human T-Lymphotropic Virus, and Human ImmunodeficiencyViruses (HIV). In a preferred aspect, the retrovirus is HIV. HIV can beany strain, form, subtype or variation in the HIV family. HIV virusesinclude, but are not limited to, HIV-I, HIV-II, HIV-III (also known asHTLV-II, LAV-I, LAV-2), and the like.

The compounds can also be used as adjunct therapy in combination withexisting therapies in the management of the aforementioned types ofviral infections. In such situations, it is preferably to administer theactive ingredients to a patient in a manner that optimizes effects uponviruses, including mutated, multi-drug resistant viruses, whileminimizing effects upon normal cell types. While this is primarilyaccomplished by virtue of the behavior of the compounds themselves, thiscan also be accomplished by targeted drug delivery and/or by adjustingthe dosage such that a desired effect is obtained without meeting thethreshold dosage required to achieve significant side effects.

Retroviruses whose infection can be treated or prevented using theinhibitors described herein include any viruses having RNA as theirprimary genetic material and use reverse transcription to produce DNA.Such viruses include, but are not limited to, Feline ImmunodeficiencyVirus (FIV), Simian Immunodeficiency Virus (SIV), Avian Leucosis Virus,Feline Leukemia Virus, Walleye Dermal Sarcoma Virus, HumanT-Lymphotropic Virus, and Human Immunodeficiency Viruses (HIV). In apreferred aspect, the retrovirus is HIV. HIV can be any strain, form,subtype or variation in the HIV family. HIV viruses include, but are notlimited to, HIV-I, HIV-II, HIV-III (also known as HTLV-II, LAV-I,LAV-2), mutated versions thereof, and the like.

Inhibitors of HIV are also active against the hepatitis B virus (HBV),and can be used in methods of treating and/or preventing HBV infection,and pharmaceutical compositions intended for this use.

IV. Pharmaceutical Compositions

The inhibitory compounds as described herein can be incorporated intopharmaceutical compositions and used to treat or prevent a viralinfection, such as a retroviral infection. The pharmaceuticalcompositions described herein include the inhibitory compounds asdescribed herein, and a pharmaceutically acceptable carrier and/orexcipient.

The manner in which the compounds are administered can vary. Thecompositions are preferably administered orally (e.g., in liquid formwithin a solvent such as an aqueous or non-aqueous liquid, or within asolid carrier). Preferred compositions for oral administration includepills, tablets, capsules, caplets, syrups, and solutions, including hardgelatin capsules and time-release capsules. Compositions may beformulated in unit dose form, or in multiple or subunit doses. Preferredcompositions are in liquid or semisolid form. Compositions including aliquid pharmaceutically inert carrier such as water or otherpharmaceutically compatible liquids or semisolids may be used. The useof such liquids and semisolids is well known to those of skill in theart.

The compositions can also be administered via injection, i.e.,intraveneously, intramuscularly, subcutaneously, intraperitoneally,intraarterially, intrathecally; and intracerebroventricularly.Intravenous administration is a preferred method of injection. Suitablecarriers for injection are well known to those of skill in the art, andinclude 5% dextrose solutions, saline, and phosphate buffered saline.The compounds can also be administered as an infusion or injection(e.g., as a suspension or as an emulsion in a pharmaceuticallyacceptable liquid or mixture of liquids).

The formulations may also be administered using other means, forexample, rectal administration. Formulations useful for rectaladministration, such as suppositories, are well known to those of skillin the art. The compounds can also be administered by inhalation (e.g.,in the form of an aerosol either nasally or using delivery articles ofthe type set forth in U.S. Pat. No. 4,922,901 to Brooks et al., thedisclosure of which is incorporated herein in its entirety); topically(e.g., in lotion form); or transdermally (e.g., using a transdermalpatch, using technology that is commercially available from Novartis andAlza Corporation). Although it is possible to administer the compoundsin the form of a bulk active chemical, it is preferred to present eachcompound in the form of a pharmaceutical composition or formulation forefficient and effective administration.

Exemplary methods for administering such compounds will be apparent tothe skilled artisan. The usefulness of these formulations may depend onthe particular composition used and the particular subject receiving thetreatment. These formulations may contain a liquid carrier that may beoily, aqueous, emulsified or contain certain solvents suitable to themode of administration.

The compositions can be administered intermittently or at a gradual,continuous, constant or controlled rate to a warm-blooded animal (e.g.,a mammal such as a mouse, rat, cat, rabbit, dog, pig, cow, or monkey),but advantageously are administered to a human being. In addition, thetime of day and the number of times per day that the pharmaceuticalformulation is administered can vary.

Preferably, the compositions are administered such that activeingredients interact with regions where viral infections are located.The compounds described herein are very potent at treating these viralinfections.

In certain circumstances, the compounds described herein can be employedas part of a pharmaceutical composition with other compounds intended toprevent or treat a particular viral infection, i.e., combinationtherapy. In addition to effective amounts of the compounds describedherein, the pharmaceutical compositions can also include various othercomponents as additives or adjuncts.

Combination or Alternation Therapy

In one embodiment, the compounds of the invention can be employedtogether with at least one other antiviral agent, chosen from entryinhibitors, reverse transcriptase inhibitors, protease inhibitors, andimmune-based therapeutic agents.

For example, when used to treat or prevent HIV infection, the activecompound or its prodrug or pharmaceutically acceptable salt can beadministered in combination or alternation with another anti-HIV agent.In general, in combination therapy, effective dosages of two or moreagents are administered together, whereas during alternation therapy, aneffective dosage of each agent is administered serially. The dosage willdepend on absorption, inactivation and excretion rates of the drug, aswell as other factors known to those of skill in the art. It is to benoted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens and schedules should beadjusted over time according to the individual need and the professionaljudgment of the person administering or supervising the administrationof the compositions.

Combination therapy may be administered as (a) a single pharmaceuticalcomposition which comprises an inhibitory compound as described herein,at least one additional pharmaceutical agent described herein, and apharmaceutically acceptable excipient, diluent, or carrier; or (b) twoseparate pharmaceutical compositions comprising (i) a first compositioncomprising an inhibitory compound as described herein and apharmaceutically acceptable excipient, diluent, or carrier, and (ii) asecond composition comprising at least one additional pharmaceuticalagent described herein and a pharmaceutically acceptable excipient,diluent, or carrier. The pharmaceutical compositions can be administeredsimultaneously or sequentially and in any order.

In use in treating or preventing viral disease, the inhibitorycompound(s) can be administered together with at least one otherantiviral agent as part of a unitary pharmaceutical composition.Alternatively, it can be administered apart from the other antiviralagents. In this embodiment, the inhibitory compound and the at least oneother antiviral agent are administered substantially simultaneously,i.e. the compounds are administered at the same time or one after theother, so long as the compounds reach therapeutic levels for a period oftime in the blood.

Combination therapy involves administering the inhibitory compound, asdescribed herein, or a pharmaceutically acceptable salt or prodrug ofthe inhibitory compound, in combination with at least one anti-viralagent, ideally one which functions by a different mechanism than theinhibitors of viral propagation described herein.

Representative Antiviral Agents

Some antiviral agents which can be used for combination therapy includeagents that interfere with the ability of a virus to infiltrate a targetcell. The virus must go through a sequence of steps to do this,beginning with binding to a specific “receptor” molecule on the surfaceof the host cell and ending with the virus “uncoating” inside the celland releasing its contents. Viruses that have a lipid envelope must alsofuse their envelope with the target cell, or with a vesicle thattransports them into the cell, before they can uncoat.

There are two types of active agents which inhibit this stage of viralreplication. One type includes agents which mimic the virus-associatedprotein (VAP) and bind to the cellular receptors, including VAPanti-idiotypic antibodies, natural ligands of the receptor andanti-receptor antibodies, receptor anti-idiotypic antibodies, extraneousreceptor and synthetic receptor mimics. The other type includes agentswhich inhibit viral entry, for example, when the virus attaches to andenters the host cell. For example, a number of “entry-inhibiting” or“entry-blocking” drugs are being developed to fight HIV, which targetsthe immune system white blood cells known as “helper T cells”, andidentifies these target cells through T-cell surface receptorsdesignated “CRX4” and “CCR5”. Thus, CRX4 and CCR5 receptor inhibitorssuch as amantadine and rimantadine, can be used to inhibit viralinfection, such as HIV, influenza, and hepatitis B and C viralinfections. Another entry-blocker is pleconaril, which works againstrhinoviruses, which cause the common cold, by blocking a pocket on thesurface of the virus that controls the uncoating process.

Further antiviral agents that can be used in combination with theinhibitory compounds described herein include agents which interferewith viral processes that synthesize virus components after a virusinvades a cell. Representative agents include nucleotide and nucleosideanalogues that look like the building blocks of RNA or DNA, butdeactivate the enzymes that synthesize the RNA or DNA once the analogueis incorporated. Acyclovir is a nucleoside analogue, and is effectiveagainst herpes virus infections. Zidovudine (AZT), 3TC, FTC, and othernucleoside reverse transcriptase inhibitors (NRTI), as well asnon-nucleoside reverse transcriptase inhibitors (NNRTI), can also beused. Integrase inhibitors can also be used.

Once a virus genome becomes operational in a host cell, it thengenerates messenger RNA (mRNA) molecules that direct the synthesis ofviral proteins. Production of mRNA is initiated by proteins known astranscription factors, and certain active agents block attachment oftranscription factors to viral DNA.

Other active agents include antisense oligonucleotides and ribozymes(enzymes which cut apart viral RNA or DNA at selected sites).

Some viruses, such as HIV, include protease enzymes, which cut viralprotein chains apart so they can be assembled into their finalconfiguration. Protease inhibitors are another type of antiviral agentthat can be used in combination with the inhibitory compounds describedherein.

The final stage in the life cycle of a virus is the release of completedviruses from the host cell. Some active agents, such as zanamivir(Relenza) and oseltamivir (Tamiflu) treat influenza by preventing therelease of viral particles by blocking a molecule named neuraminidasethat is found on the surface of flu viruses.

Still other active agents function by stimulating the patient's immunesystem. Interferons, including pegylated interferons, are representativecompounds of this class. Interferon alpha is used, for example, to treathepatitis B and C. Various antibodies, including monoclonal antibodies,can also be used to target viruses.

Any of the above-mentioned compounds can be used in combination therapywith the inhibitors described herein.

The appropriate dose of the compound is that amount effective to preventoccurrence of the symptoms of the disorder or to treat some symptoms ofthe disorder from which the patient suffers. By “effective amount”,“therapeutic amount” or “effective dose” is meant that amount sufficientto elicit the desired pharmacological or therapeutic effects, thusresulting in effective prevention or treatment of the disorder.

When treating viral infections, an effective amount of the inhibitorycompound is an amount sufficient to suppress the growth andproliferation of the virus. Viral infections can be prevented, eitherinitially, or from re-occurring, by administering the compoundsdescribed herein in a prophylactic manner. Preferably, the effectiveamount is sufficient to obtain the desired result, but insufficient tocause appreciable side effects.

The effective dose can vary, depending upon factors such as thecondition of the patient, the severity of the viral infection, and themanner in which the pharmaceutical composition is administered. Theeffective dose of compounds will of course differ from patient topatient, but in general includes amounts starting where desiredtherapeutic effects occur but below the amount where significant sideeffects are observed.

The compounds, when employed in effective amounts in accordance with themethod described herein, are effective at inhibiting the proliferationof certain viruses, but do not significantly effect normal cells.

For human patients, the effective dose of typical compounds generallyrequires administering the compound in an amount of at least about 1,often at least about 10, and frequently at least about 25 μg/24hr/patient. The effective dose generally does not exceed about 500,often does not exceed about 400, and frequently does not exceed about300 μg/24 hr/patient. In addition, administration of the effective doseis such that the concentration of the compound within the plasma of thepatient normally does not exceed 500 ng/mL and frequently does notexceed 100 ng/mL.

V. Methods for Identifying an Inhibitor of Retroviral Propogation

The compounds described herein can be evaluated for their ability toinhibit viral propagation, for example, retroviral propagation, usingthe methods described herein. The retroviral propagation can beinhibited, for example, by

a) inhibiting retroviral reverse transcription,

b) inhibiting the binding of a host cell tRNA and a target nucleic acidmolecule,

c) inhibiting the viruses recruitment of the retroviral primer, humantRNA^(Lys3),

d) inhibiting HIV translation of viral RNA to precursor proteins, and/or

e) inhibiting HIV's final packaging and assembly.

These individual methods for identifying inhibitors of retroviralpropagation are discussed below.

Identifying Inhibitors of Retroviral Reverse Transcription

In one aspect, putative inhibitors of retroviral reverse transcriptioncan be identified. In another aspect, putative inhibitors of tRNA'sability to bind to a target nucleic acid molecule can be identified. Theidentification can be done in in a high through-put manner. Transfer RNA(tRNA) is involved in reverse transcription through the recognition of acorresponding site on the retroviral genome priming reversetranscription. Identifying inhibitors of reverse transcription may leadto the identification of therapeutic compounds for use in treatingretroviral infection in a host cell.

The screening methods involve forming a mixture having a tRNA anticodonstem-loop (ASL) fragment, a target nucleic acid molecule that is capableof binding to the tRNA fragment, and a test compound. In one aspect, thetarget nucleic acid molecule corresponds to a fragment of the retroviralgenome involved in reverse transcription. The resulting mixture isincubated under conditions that allow binding of the tRNA fragment andthe target nucleic acid in the absence of the test compound. The methodfurther involves detecting whether the test compound inhibits thebinding of the tRNA fragment to the target nucleic acid, where theabsence of binding of the tRNA ASL fragment and the target nucleic acidmolecule is indicative of the test compound being an inhibitor ofretroviral reverse transcription. In one aspect, the detection involvesthe use of labels to detect the inhibition of binding of the tRNAfragment to the target nucleic acid molecule.

Methods for Identifying Inhibitors of Binding of a Host Cell tRNA to aTarget Nucleic Acid Molecule

In another aspect, the ability of a putative inhibitor to bind a tRNA toa target nucleic acid molecule can be assayed. The assay involvesforming a mixture containing a host cell tRNA ASL fragment, a targetnucleic acid molecule that is capable of binding to the tRNA fragment,and a test compound. The resulting mixture is incubated under conditionsthat allow binding of the tRNA fragment and the target nucleic acid inthe absence of the test compound. The method further involves detectingwhether the test compound inhibits the binding of the tRNA fragment tothe target nucleic acid, where binding of the tRNA ASL fragment and thetarget nucleic acid molecule is indicative of the test compound being aninhibitor of binding of a tRNA to a target nucleic acid molecule. In oneaspect, the detection involves the use of labels to detect theinhibition of binding of the tRNA fragment to the target nucleic acidmolecule.

Methods for Identifying Inhibitors of HIV Reverse Transcription (RT)Complex Formation

In another aspect, the ability of a compound to function as an inhibitorof HIV reverse transcriptase (RT) complex formation can be assayed. Theassay involves forming a mixture containing a tRNA ASL fragment, atarget nucleic acid molecule capable of binding to the tRNA fragment,and a test compound. The resulting mixture is incubated under conditionsthat allow binding of the tRNA fragment and the target nucleic acid inthe absence of the test compound. The method further involves detectingwhether the test compound inhibits the binding of the tRNA fragment tothe target nucleic acid. In one aspect, the detection involves the useof labels to detect the inhibition of binding of the tRNA fragment tothe target nucleic acid molecule, where the inhibition indicates thatthe test compound is capable of inhibiting the formation of the RTcomplex.

In another aspect, the assay may involve detecting the binding of theputative inhibitor to either the tRNA fragment, the target nucleic acid,or both the tRNA fragment and the target nucleic acid. In one aspect,the binding of the putative inhibitor is indicative of the test compoundbeing an inhibitor of retroviral propagation, retroviral infection,reverse transcription, or tRNA binding.

Methods for Identifying Inhibitors of Viral Recruitment of HumantRNA^(Lys3).

In yet another aspect, the ability of a putative inhibitor to inhibitHIV's recruitment of the retroviral primer, human tRNA^(Lys3) can beassayed. The assay involves forming a mixture comprising a linearsequence of a tRNA anticodon stem loop fragment that is not capable offorming a stem-loop, a target nucleic acid molecule capable of bindingto the tRNA anticodon stem loop fragment, and a test compound, whereinthe target nucleic acid molecule corresponds to a portion of aretroviral genome involved in recruitment of retroviral primerrecruitment. The mixture is incubated under conditions that allowbinding of the tRNA anticodon stem loop fragment and the target nucleicacid molecule in the absence of the test compound. One can then detectwhether or not the test compound inhibits the binding of the tRNAanticodon stem loop fragment and the target nucleic acid molecule. Theabsence of binding of the tRNA ASL fragment and the target nucleic acidmolecule is indicative of the test compound being an inhibitorretroviral primer recruitment.

Methods for Identifying Inhibitors of Viral RNA Translation

In still another aspect, a ability of a putative inhibitor of viral RNAtranslation to viral precursor proteins can be assayed. The assayinvolves forming a mixture comprising a linear sequence of a tRNAanticodon stem loop fragment that is not capable of forming a stem-loop,a target nucleic acid molecule capable of binding to the tRNA anticodonstem loop fragment, and a test compound; incubating the mixture underconditions that allow binding of the tRNA anticodon stem loop fragmentand the target nucleic acid molecule in the absence of the testcompound; and detecting whether or not the test compound inhibits thebinding of the tRNA fragment and the target nucleic acid molecule wherebinding of the tRNA ASL fragment and the target nucleic acid molecule isindicative of the test compound being an inhibitor of tRNA recruitmentduring viral RNA translation to viral precursor proteins.

The inhibitors can inhibit the retroviral infection by inhibiting anystep of a virus lifecycle, including, but not limited to, reversetranscription, viral assembly, RT complex formation, recruitment of theretroviral primer, human tRNA^(Lys3), translation of viral RNA toprecursor proteins, and the final packaging and assembly. Moreover, theinhibitors may inhibit retroviral infection, delay the infection, orslow the progression of the infection.

VI. tRNA Fragments Useful in the Methods Described Herein

The tRNA fragments (or “tool tRNA fragments”) for use in the screeningmethods described herein can be a fragment from any tRNA. Specific tRNAfragments described in the formulas below are another aspect of theinvention, and these fragments can be included in the kits describedherein.

The tRNA fragments (or “tool tRNA fragments”) for use in the methods ofthe present disclosure can be a fragment from any tRNA. The tRNAfragment may be obtained or derived from or corresponds to a tRNA^(Ala),tRNA^(Arg), tRNA^(Asn), tRNA^(Asp), tRNA^(Cys), tRNA^(Gln), tRNA^(Glu),tRNA^(Gly), tRNA^(His), tRNA^(Ile), tRNA^(Leu), tRNA^(Lys), tRNA^(Met),tRNA^(Phe), tRNA^(Pro), tRNA^(Ser), tRNA^(Thr), tRNA^(Trp), tRNA^(Tyr),and tRNA^(Val). In one aspect, the tRNA fragment corresponds totRNA^(Lys). In another aspect, the tRNA fragment is derived from orcorresponds to the tRNA^(Lys) anticodon stem loop (ASL). In anotheraspect, the tRNA fragment corresponds to a fragment of nucleotides 32-43of the human tRNA^(Lys). The position numbers used herein refer to thenucleotide position numbering of the conventional tRNA numbering asdisclosed in Sprinzl, et al. Nucl. Acids. Res., 26, 148-153 (1998). Inone aspect, the tRNA fragment is a fragment from a host cell tRNA, suchas a mammalian host cell, including, but not limited to, human, feline,and simian host cells.

The tRNA fragments may incorporate one or more modified nucleosides. Inone aspect, the tRNA fragment incorporates one, two, three, or moremodified nucleosides into the nucleic acid sequence. In another aspect,the tRNA fragments incorporate three modified nucleosides into the tRNAfragment nucleic acid molecules. Modified nucleosides that can beincorporated into the tRNA fragments include any modified nucleotide,including, but not limited to unknown modified adenosine (?A),1-methyladenosine (m1A), 2-methyladenosine (m2A),N⁶-isopentenyladenosine (i6A), 2-methylthio-N⁶-isopentenyladenosine(ms2i6A), N⁶-methyladenosine (m6A), N⁶-threonylcarbamoyladenosine (t6A),N⁶-methyl-N⁶ threonylcarbomoyladenosine (m6t6A),2-methylthio-N⁶-threonylcarbamoyladenosine (ms2t6A),2′-O-methyladenosine I Inosine (Am), 1-methylinosine Ar(p)2′-O-(5-phospho)ribosyladenosine (m1I),N⁶-(cis-hydroxyisopentenyl)adenosine (io6A), Unknown modified cytidine(?C), 2-thiocytidine (s2C), 2′-O-methylcytidine (Cm), N⁴-acetylcytidine(ac4C), 5-methylcytidine (m5C), 3-methylcytidine (m3C), lysidine (k2C),5-formylcytidin (f5C), 2′-O-methyl-5-formylcytidin (f5Cm), unknownmodified guanosine (?G), 2′-O-(5phospho)ribosylguanosine (Gr(p)),1-methylguanosine (m1G), N²-methylguanosine (m2G), 2′-O-methylguanosine(Gm), N²N²-dimethylguanosine (m22G), N²,N²,2′-O-trimethylguanosine(m22Gm), 7-methylguanosine (m7G), archaeosine (fa7d7G), queuosine (Q),mannosyl-queuosine (manQ), galactosyl-queuosine (galQ), wybutosine (yW),peroxywybutosine (02yW), unknown modified uridine (?U),5-methylaminomethyluridine (mnm5U), 2-thiouridine (s2U),2′-O-methyluridine (Um), 4-thiouridine (s4U), 5carbamoylmethyluridine(ncm5U), 5-methoxycarbonylmethyluridine (mcm5U),5methylaminomethyl-2-thiouridine (mnm5 s2U),5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), uridine 5-oxyaceticacid (cmo5U), 5-methoxyuridine (mo5U), 5carboxymethylaminomethyluridine(cmnm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U),3-(3-amino-3-carboxypropyl)uridine (acp3U),5-(carboxyhydroxymethyl)uridinemethyl ester (mchm5U),5-carboxymethylaminomethyl-2′-O-methyluridine (cmnm5Um),5-carbamoylmethyl-2′-O-methyluridine (ncm5Um), Dihydrouridine (D),pseudouridine (ψ), 1-methylpseudouridine (mlψ), 2′-O-methylpseudouridine(ψm), ribosylthymine (m5U), 5-methyl-2-thiouridine (m5s2U), and5,2′-O-dimethyluridine (m5Um).

In a preferred aspect, the fragment tRNA contains modified nucleic acidscorresponding to positions 34, 37, and 39 in the anticodon stem loop ofa tRNA. The position numbers used herein refer to the nucleotideposition numbering of the conventional tRNA numbering as disclosed inSprinzl, et al. Nucl. Acids. Res., 26, 148-153 (1998). In one aspect,the tRNA fragment comprises, or consists of, a molecule having thesequence 5′-GCUXUUAYZCUG (SEQ ID NO. 1), in which the X, Y, and Z referto modified or unmodified nucleosides. In one aspect, the X, Y, and Zrefer to modified nucleosides, such as mnm5s2U, mcm5s2U, ms2t6A, s2U, ψ,and t6A. In another aspect, the tRNA fragment has the nucleic acidsequence 5′-CU(mnm5s2U)UU(ms2t6A)A(ψ)CUGC (SEQ ID NO. 2). In anotheraspect, the tRNA fragment has the nucleic acid sequence5′-GCU(mnm5s2U)UU(ms2t6A)A(ψ)CUG (SEQ ID NO. 3).

The tRNA fragment may correspond to any portion of the tRNA involved inpropagation of the retrovirus through binding, directly or indirectly,to the retroviral genome. In a preferred aspect, the tRNA fragmentcorresponds to the anticodon stem loop (ASL) of the tRNA.

The tRNA fragment may correspond to any portion of the host cell's tRNAinvolved in nucleotide binding, such as involvement in the reversetranscription (RT) complex formation. For example, the tRNA may beinvolved in binding to a retroviral genome to initiate, prime, orfacilitate reverse transcription of the retroviral genome. In oneaspect, the fragment tRNA corresponds to a fragment of the anticodonstem loop of any tRNA. In one aspect, the fragment corresponds to afragment from the anticodon stem loop of tRNA^(−Lys). In another aspect,the tRNA fragment corresponds to a fragment from the anticodon stem loopof human tRNA^(−Lys). In another aspect, the tRNA fragment correspondsto a fragment from nucleotides 32-43 of human tRNA^(Lys3).

The tRNA fragment may also be any length of a fragment from a tRNA. Inone aspect, the tRNA fragment comprises a fragment of between 9 to 15continuous nucleotides of a tRNA, 10 to 14 continuous nucleotides of atRNA, or between 11 to 13 continuous nucleotides of a tRNA. In anotheraspect, the fragment is a fragment of 8, 9, 10, 11, 12, 13, 14, 15, or16 continuous nucleotides of a tRNA. In a further aspect, the fragmentis a fragment of 12 continuous nucleotides of a tRNA.

The tRNA fragment may or may not be capable of forming a secondarystructure. In a one aspect, the tRNA fragment is not capable of forminga stem loop structure with itself. In another aspect, the fragment is alinear fragment of a tRNA that is not capable of forming a stem loopstructure with itself.

The tRNA fragment may also be linked to additional nucleic acids. Forexample, the tRNA fragment may be linked to one or more additionalnucleic acids depending on the assay method. In one aspect, the tRNAfragment may be linked to nucleotides used to attach the fragment to asolid support surface. In another aspect, the fragment tRNA is linked toadditional nucleic acid molecules at one or both terminal end of thetRNA fragment. In another aspect, the fragment tRNA is linked toadditional nucleic acid molecules at both terminal ends. The additionalnucleic acid sequences can be any length, preferably between 8 and 16nucleotides, between 10 and 14 nucleotides, more preferably 12nucleotides in length. In one aspect, the terminal sequences do notallow the tRNA fragment to form a secondary structure, such as a hairpinloop structure.

A target nucleic acid molecule may correspond to any nucleic acidmolecule, such as a DNA or an RNA molecule that is involved inretroviral propagation or retroviral reverse transcription. In oneaspect, the target nucleic acid molecule corresponds to any nucleic acidmolecule that is capable of binding to the tRNA fragment and is involvedin retroviral propagation or reverse transcription. In another aspect,the target nucleic acid molecule corresponds to a nucleic acid moleculeinvolved in reverse transcription of a retroviral genome. In anotheraspect, the target nucleic acid molecule corresponds to ribonucleic acidfrom a retroviral genome. In another aspect, the target nucleic acidmolecule corresponds to a nucleic acid molecule that is involved inpriming retroviral reverse transcription.

The target nucleic acid molecule may be any length and may include theentire retroviral genome and fragments thereof. In one aspect, thetarget nucleic acid molecule includes any fragment of a retroviralgenome involved in tRNA binding, and includes fragments of at least 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or morenucleotides. In another aspect, the target nucleic acid is about thesame, or is the same length as the tool tRNA fragment.

In another aspect, the target nucleic acid molecule corresponds to anucleic acid molecule from a Human Immunodeficiency Virus (HIV), such asHIV-1 or HIV-2. In another aspect, the target molecule corresponds toHIV-1. In another aspect, the target nucleic acid molecule correspondsto a nucleic acid molecule involved in priming HIV reversetranscription.

Such target nucleic acid molecules can be derived from or correspond toany portion of the HIV genome involved in reverse transcription throughthe binding or association with a host cell tRNA. In one aspect, thetarget nucleic acid molecule is derived from or corresponds to the 5′untranslated region of the HIV genome. In another aspect, the targetnucleic acid molecule corresponds to a fragment including residues 157to 169 of the 5′ untranslated region of HIV-1. The target nucleic acidsequence may be complementary to the tRNA fragment. In a one aspect, thetarget nucleic acid molecule comprises the nucleic acid sequence5′-GCGGUGUAAAAG (SEQ ID NO. 4).

Specific Isolated tRNA Fragments

In one aspect, the isolated tRNA fragment comprises the sequence5′-GCUXUUAYZCUG (SEQ ID NO. 1), in which the X, Y, and Z refer tomodified nucleosides.

Representative modified nucleosides include unknown modified adenosine(?A), 1-methyladenosine (m1A), 2-methyladenosine (m2A),N⁶-isopentenyladenosine (i6A), 2-methylthio-N⁶-isopentenyladenosine(ms2i6A), N⁶-methyladenosine (m6A), N⁶-threonylcarbamoyladenosine (t6A),N⁶-methyl-N⁶ threonylcarbomoyladenosine (m6t6A),2-methylthio-N⁶-threonylcarbamoyladenosine (ms2t6A),2′-O-methyladenosine I Inosine (Am), 1-methylinosine Ar(p)2′-O-(5-phospho)ribosyladenosine (m1I),N⁶-(cis-hydroxyisopentenyl)adenosine (io6A), Unknown modified cytidine(?C), 2-thiocytidine (s2C), 2′-O-methylcytidine (Cm), N⁴-acetylcytidine(ac4C), 5-methylcytidine (m5C), 3-methylcytidine (m3C), lysidine (k2C),5-formylcytidin (f5C), 2′-O-methyl-5-formylcytidin (f5Cm), unknownmodified guanosine (?G), 2′-O-(5phospho)ribosylguanosine (Gr(p)),1-methylguanosine (m1G), N²-methylguanosine (m2G), 2′-O-methylguanosine(Gm), N²N²-dimethylguanosine (m22G), N²,N²,2′-O-trimethylguanosine(m22Gm), 7-methylguanosine (m7G), archaeosine (fa7d7G), queuosine (Q),mannosyl-queuosine (manQ), galactosyl-queuosine (galQ), wybutosine (yW),peroxywybutosine (02yW), unknown modified uridine (?U),5-methylaminomethyluridine (mnm5U), 2-thiouridine (s2U),2′-O-methyluridine (Um), 4-thiouridine (s4U), 5carbamoylmethyluridine(ncm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5methylaminomethyl-2-thiouridine (mnm5 s2U),5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), uridine 5-oxyaceticacid (cmo5U), 5-methoxyuridine (mo5U), 5carboxymethylaminomethyluridine(cmnm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5 s2U),3-(3-amino-3-carboxypropyl)uridine (acp3U),5-(carboxyhydroxymethyl)uridinemethyl ester (mchm5U),5-carboxymethylaminomethyl-2′-O-methyluridine (cmnm5Um),5-carbamoylmethyl-2′-O-methyluridine (ncm5Um), Dihydrouridine (D),pseudouridine (ψ), 1-methylpseudouridine (mlψ), 2′-O-methylpseudouridine(ψm), ribosylthymine (m5U), 5-methyl-2-thiouridine (m5 s2U), and5,2′-O-dimethyluridine (m5Um).

In one embodiment, the modified nucleosides are mnm5s2U, mcm5s2U,ms2t6A, s2U, ψ, or t6A.

One specific tRNA fragment comprises the nucleic acid sequence5′-CU(mnm5 s2U)UU(ms2t6A)A(‘l’)CUGC.

Another specific tRNA fragment comprises the nucleic acid sequence5′-GCU(mnm5 s2U)UU(ms2t6A)A(‘l’)CUG.

Any of these tRNA fragments can further comprise a label. The label canbe detectable, either directly or indirectly, by spectroscopic,photochemical, biochemical, immunochemical, or chemical means.

Representative labels include radioactive isotopes (for example, ³²P,³⁵S, and ³H), dyes, fluorescent dyes (for example, Cy5 and Cy3),fluorophores (for example, fluorescein), electron-dense reagents,enzymes and their substrates (for example, as commonly used inenzyme-linked immunoassays, such as, alkaline phosphatase and horseradish peroxidase), biotin-streptavidin, digoxigenin, or hapten; andproteins for which antisera or monoclonal antibodies are available. Thelabel can also be an “affinity tag.”

Where the label comprises an affinity tag, the isolated tRNA fragmentscan be captured with a complimentary ligand coupled to a solid supportthat allows for the capture of the affinity tag-labeled tRNA fragment.Representative affinity tags and complimentary partners includebiotin-streptavidin, complimentary nucleic acid fragments (for example,oligo dT-oligo dA, oligo T-oligo A, oligo dO-oligo dC, oligo O-oligo C),aptamers, or haptens and proteins for which antisera or monoclonalantibodies are available.

When a biological interaction brings the beads together, a cascade ofchemical reactions acts to produce a greatly amplified signal. On laserexcitation, a photosensitizer in the “Donor” bead converts ambientoxygen to a more excited singlet state. The singlet state oxygenmolecules diffuse across to react with a thioxene derivative in theAcceptor bead generating chemiluminescence at 370 nm that furtheractivates fluorophores contained in the same bead. The fluorophoressubsequently emit light at 520-620 nm.

In one example of a commercially-available alpha bead, the Donor beadscomprise biotin or are directly bound to RNA. The Acceptor beads includea His6 tag, hemagglutinin (HA), digoxin/digoxigenin (DIG), orfluorescein (FITC).

VII. Synthetic Methods for Producing Isolated Ribonucleotides

A variety of methods are known in the art for making nucleic acidshaving a particular sequence or that contain particular nucleic acidbases, sugars, internucleoside linkages, chemical moieties, and othercompositions and characteristics. Anyone or any combination of thesemethods can be used to make a nucleic acid, polynucleotide, oroligonucleotide for the present invention. Said methods include, but arenot limited to: (1) chemical synthesis (usually, but not always, using anucleic acid synthesizer instrument); (2) post-synthesis chemicalmodification or derivatization; (3) cloning of a naturally occurring orsynthetic nucleic acid in a nucleic acid cloning vector (e.g., seeSambrook, et aI., Molecular Cloning: A Laboratory Approach 2nd ed., ColdSpring Harbor Laboratory Press, 1989) such as, but not limited to aplasmid, bacteriophage (e.g., mB or lamda), phagemid, cosmid, fosmid,YAC, or BAC cloning vector, including vectors for producingsingle-stranded DNA; (4) primer extension using an enzyme with DNAtemplate-dependent DNA polymerase activity, such as, but not limited to,Klenow, T4, T7, rBst, Taq, Tfl, or Tth DNA polymerases, includingmutated, truncated (e.g., exo-minus), or chemically-modified forms ofsuch enzymes; (5) PCR (e.g., see Dieffenbach, C. W., and Dveksler, eds.,PCR Primer: A Laboratory Manual, 1995, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.); (6) reverse transcription (includingboth isothermal synthesis and RT-PCR) using an enzyme with reversetranscriptase activity, such as, but not limited to, reversetranscriptases derived from avian myeloblasosis virus (AMV), Maloneymurine leukemia virus (MMLV), Bacillus stearothermophilus (rBst),Thermus thermophilus (Tth); (7) in vitro transcription using an enzymewith RNA polymerase activity, such as, but not limited to, SP6, T3, orT7 RNA polymerase, Tth RNA polymerase, E. coli RNA polymerase, oranother enzyme; (8) use of restriction enzymes and/or modifying enzymes,including, but not limited to exo- or endonucleases, kinases, ligases,phosphatases, methylases, glycosylases, terminal transferases, includingkits containing such modifying enzymes and other reagents for makingparticular modifications in nucleic acids; (9) use of polynucleotidephosphorylases to make new randomized nucleic acids; (10) othercompositions, such as, but not limited to, a ribozyme ligase to join RNAmolecules; and/or (11) any combination of any of the above or othertechniques known in the art. Oligonucleotides and polynucleotides,including chimeric (i.e., composite) molecules and oligonucleotides withnon-naturally-occurring bases, sugars, and internucleoside linkages arecommercially available (e.g., see the 2000 Product and Service Catalog,TriLink Biotechnologies, San Diego, Calif., USA)

The tRNA fragment or the target nucleic acid, or both the tRNA fragmentand the target nucleic acid molecule may be detectably labeled tofacilitate detection. In a preferred aspect, the tRNA fragment islabeled with a fluorophore to facilitate detection. In another aspect,the target nucleic acid molecule is labeled with biotin to facilitatedetection. In another preferred aspect, the tRNA fragment is labeledwith a fluorophore and the target nucleic acid molecule is labeled withbiotin.

The tRNA fragment and target nucleic acid molecule may be labeled, forexample, at either the 5′ terminus, the 3′-terminus, or combinations ofthe 5′-terminus and the 3′terminus to facilitate detection. In addition,the test compound may also be labeled. In another embodiment, the tRNAfragment and the target nucleic acid molecule may have a detectablelabel attached to an internal position of the molecule to facilitatedetection.

VIII. Methods for Detecting Binding (or Inhibition Thereof) of TargetRNA to tRNA

The methods for detecting binding of the target RNA to the tRNA or theinhibition of such binding may be performed using any method for suchdetection. For example, the AlphaScreen® assay (Packard InstrumentCompany, Meriden, Conn.). AlphaScreen® technology is an “AmplifiedLuminescent Proximity Homogeneous Assay” method utilizing latexmicrobeads (250 nm diameter) containing a photosensitizer (donor beads),or chemiluminescent groups and fluorescent acceptor molecules (acceptorbeads). Upon illumination with laser light at 680 nm, thephotosensitizer in the donor bead converts ambient oxygen tosinglet-state oxygen. The excited singlet-state oxygen molecules diffuseapproximately 250 nm (one bead diameter) before rapidly decaying. If theacceptor bead is in close proximity to the donor bead (i.e., by virtueof the interaction of the target RNA and tRNA fragment), thesinglet-state oxygen molecules reacts with chemiluminescent groups inthe acceptor beads, which immediately transfer energy to fluorescentacceptors in the same bead. These fluorescent acceptors shift theemission wavelength to 520-620 nm, resulting in a detectable signal.Antagonists of the interaction of the target RNA with the tRNA fragmentwill thus inhibit the shift in emission wavelength, whereas agonists ofthis interaction would enhance it.

The disclosed methods may be performed by mixing the componentnucleotide (e.g. the tool tRNA and the target RNA) and the test compoundin any order, orsimultaneously. For example, a target RNA may be firstcombined with a test compound to form a first mixture, and then a tooltRNA fragment may be added to form a second mixture. In another example,a target RNA, a tool tRNA and the test compound may all be mixed at thesame time before incubation. In one aspect, the mixture is incubatedunder conditions that allow binding of the tRNA fragment and the targetnucleic acid in the absence of the test compound.

The inhibition of binding of the tRNA fragment and the target nucleicacid molecule by the test compound may be detected using any methodavailable for the detection of inhibition. In one aspect, thedetermining step may be performed using methods including, but notlimited to, gel shift assays, chemical and enzymatic footprinting,circular dichroism and NMR spectroscopy, equilibrium dialysis, or in anyof the binding detection mechanisms commonly employed with combinatoriallibraries of probes or test compounds. The inhibition of bindingindicates that the test compound may be useful for inhibitingpropagation of the virus in the host.

The invention will be further explained by the following illustrativeexamples, which are intended to be non-limiting.

EXAMPLES Example 1 Synthesis of Linear tRNA Anticodon Stem LoopSequences

The first step in producing the fragment tRNA anticodon stem loop (ASL)sequences is the synthesis of the modified nucleotides, also known asphosphoramidites (Agris et. al Biochimie. (1995) 77(1-2):125-34). Themodified nucleotides are then used during the synthesis of the RNAoligomers (Ogilvie et. al. Proc Natl Acad Sci USA. (1988) 85:5764-8).Synthetic approaches overcome the substantial barrier of obtainingsufficient amounts of natural products for the functionalcharacterization studies. In addition to providing the fully modifiedASL for characterization of the fragment tRNA:target nucleotide binding,the synthetic approach allows for the preparation of intermediatesteps/forms of the modified material that can further elucidate theindividual contribution of each modification step in enhanced tRNAbinding.

Modified base nucleic acid molecules were prepared using a combinationof methods for the synthesis, incorporation, and purification of all themodified nucleotides found in the ASL^(Lys3) human tRNA. Modified basephosphoramidites were prepared using known methods, such as thosedisclosed in Ogilive et. aI., 1988. The ASL^(Lys3) contains 3 modifiedbases denoted as mcm5s2U, ms2t6A and pseudouridine. Synthesis of thephosphoramides needed for the preparation of the synthetic mimics isdescribed below in detail. Protocols for the polymers synthesis followthose developed for automated RNA synthesis (Ogilive et. al., 1988) withvariations specific to the synthesis of the ASL^(Lys3) mimics describedbelow. The description includes methods for the removal of protectiongroup required for automated synthesis and purification of the finalproducts used in the assay.

The protecting group is subsequently removed after synthesis of the RNAoligomer. The addition of a protecting group to each modified base andribose is described. While 2 position thio-groups can be oxidized instandard RNA synthesis protocols this has been overcome by using thetert-butyl hydroperoxide (10% solution in acetonitrile) oxidizing agent(Kumar and Davis, 1997). These synthetic RNA oligomers have been used inboth functional (Yarian 2002 and Phelps 2004) and structural studies(Stuart 2000 and Murphy 2004).

Example IA: The Synthesis of the Protected Monomer Phosphoramiditesmcm5s2U

The mcm5s2U nucleoside was prepared following published methods (Reeseand Sanghvi 1984). Briefly, 2 thiouridine was heated with 5 molarequivalents each of pyrrolidine and formaldehyde in aqueous solution for1 h, under reflux, resulting in2′,3′-0isopropylidene-5-pyrrolidinomethyl-2-thiouridine. This base wassubsequently treated with 10 molar equivalents of methyl iodide inacetonitrile at room temperature. After 16 hours, the products wereconcentrated under reduced pressure to give the putative methiodidewhich was then dissolved in acetonitrile and allowed to react with 3molar equivalents of glycine t-butyl ester' at room temperature for 16h. This product was then purified and protection of the ribose andphosphitylation follow the general scheme described below.

ms2t6A

The monomer was obtained by condensation of the 2′,3′,5′-O-triacetylderivative of ms2A with the isocyanate derived fromL-threonini-O-t-butyldimethylsilyl (TBDMS)-pnitrophenylethyl ester,under conditions which eliminate racemization of the amino acid. Theproduct was selectively deprotected at the sugar moiety. Standardprocedures were employed for final protection of the 5′-O- and2′-O-functions with dimethoxytrityl (DMTr) and with TBDMS groups,respectively, as well as for 3′-O-phosphitylation (Agris et aI., 1995).

S2U

The thio group was not protected in this synthesis. Protection of theribose and phosphitylation follow the general scheme in panel C ofFIG. 1. Protection of the ribose and phosphitylation follow the generalscheme described below.

The sugar-protected phenyl carbamate 6 of t6A nucleoside was synthesizedfrom 1-O-acetyl-2,3,5-tri-O-benzoylribofuranose The carbamate wastreated with L-threonine to furnish the sugar-protected t6A nucleosideusing the method of Hong and Chheda The remaining synthetictransformations followed general scheme described below.

Example IB: General Procedure for Ribose Protection and Phosphitylation

Methods for the protection of the modified nucleotide bases prior tosynthesis of the RNA oligomer are provided (FIG. 1). Panel A of FIG. 1illustrates protection with trifluoryl acetic acid. Panel B illustratesprotection with benzoyl, and panel C illustrates the general protectionof the ribose hydroxyl groups.

After base protection the scheme for the synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-O-tertbutyidimethylsilyi-modifiedribonucleoside-3′-O-(2-cyanoethyl-N-diisopropyl)phosphoramidites is thesame for both modified nucleotides (Panel C, FIG. 1). The protectednucleoside was dried by co-evaporation twice with pyridine and dissolvedin pyridine. Tert-butyldimethylchlorosilane and imidazole were added andreacted for 4 hours at room temperature. The excess silyl chloride wasdecomposed with water and dichloromethane. The aqueous layer wasextracted twice with dichloromethane and combined with the organiclayer. The solvent was evaporated by vacuum yielding a gum which is thendissolved in ether and precipitated by pouring slowly into petroleumether (4060° C.) with stirring. The precipitate was collected and washedtwice with petroleum ether. At this point the crude product containsthree components; the 2′,3′ disilylated, 2′ silylated (major product)and 3′ silylated isomers. The pure 2′ protected isomer was purified bysilica gel column chromatography. This product is then ready forphosphitylation.

TheN-protected-5′-O-dimethoxytrityl-2′-O-tert-butyldimethylsilylribonucleosideswere dried by two co-evaporations with anhydrous pyridine and THF. Theresidue was then dissolved in anhydrous THF under argon.Dimethylaminopyridine, N,N,N-ethyldiisopropylamine andcyano-ethoxydiisopropy amino-chlorophosphine were added through a rubberseptum. After 2 hours the reaction mixture, was quenched with ethylacetate and washed with 5% sodium bicarbonate followed by water. Aqueouswashes were back extracted with ethyl acetate. Combined organic layerswere dried over sodium sulphate. Solvent was evaporated yielding aviscous oil. The product was co-evaporated twice with toluene and thepale yellow phosphoramidite products were purified by flash silica gelchromatography.

Example IC: Protocols for the Synthesis of the Modified RNA Polymers

The synthesis of the RNA followed standard protocols for a 1 mol scaleby solid phase b-cyanoethyl phosphoramidite chemistry with 2′-OTBDMSprotection (Usman et aI., 1987), and N-4-tbutyl phenoxyacetyl (tac)protection of A, G and C monomers (Sinha et aI., 1993). A, G, C and Umonomers with tac and 2′-O-TBDMS protection andrC(tac)-succinylcontrolled pore glass (CPG) support with the followingvariations. Addition of the unmodified A, C, G and U monomers werecoupled in 5-fold molar excess for 6 min in the presence of 0.3 M5-(benzylthio)-IH-tetrazole in acetonitrile (Welz and Muller, 2002),whereas mcm5s2U and ms2t6A monomers were used in 3-fold excess andcoupled for 10 min. Following the coupling, a 2 min capping wasperformed with tac anhydride and then a 3 min oxidation with 1M cumenehydroperoxide in toluene. At the end of the synthesis the5′dimethoxytrityl group was left in place.

Example ID: Protocols for the Deprotection of the Intermediates

The deprotection of the RNA was carried out in 3 steps as follows. Theargon dried CPG carrying the fully protected RNA was treated with 20 mlof absolutely anhydrous 10% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) intetrahydrofuran for 45 min at 45° C. to beliminate thep-nitrophenylethyl and 2-cyanoethyl protecting groups. The supernatantwas removed under a blanket of argon and the CPG was washed twice withdry THF. The CPG carrying partially deprotected RNA was then treatedwith 20 ml of 10% DBU in dry methanol under argon for 18 h at roomtemperature to cleave the nucleobase protecting groups and cleave theRNA from the CPG. The supernatant and methanol washings were dried in aSpeedvac in a Falcon tube and then dried for 3 days in high vacuum (10)3Torr) over phosphorus pentaoxide to remove the residual DBU. The2′-O-TBDMS protected RNA was desilylated using 12 ml of triethylaminetrihydrofluoride (Gasparutto et aI., 1992) with vigorous stirring during24 h at room temperature. During this step the DMT group is also removedfrom the 5′-terminal G residue. The reaction was quenched by addition ofsterile water (1.2 ml) and the crude RNA was precipitated with butanoland kept at 20° C. for 24 h to complete the precipitation. The RNA wascollected by centifugation, washed with butanol.

Example IE: Purification of the RNA Polymers

The synthetic RNA polymer products are purified by HPLC. The deprotectedmaterial is desalted using C18 SEP-PAK and purified by preparativeanion-exchange HPLC using a gradient of sodium chloride. In some casesadditional purification is required using reverse phase chromatography.To assure that the polymer product is correct it is analyzed byelectrospray mass spectroscopy and nucleoside composition analysis.

Example II. Inhibitor Screening Assay

Two assays were developed using tool and target RNAs, the immobilizedassay and the Alphascreen assay (FIG. 3). Both assays use the same twoRNA components (the target RNA and the tRNA fragment). In the example,the HIV viral RNA target is a 12mer with a 3′ Biotin, while the HumantRNA mimic is a synthetic 12mer containing the native modifiednucleotides and 3′ fluorescein. These two RNAs mimic an essentialcomplex of the HIV replication complex.

As set forth more fully below, the immobilization assay uses a threestep process that first involves the binding of the target RNA to anavidin coated microtiter plate. Then, the test compound (drug/smallmolecule), denoted as a star, is incubated with the target sequence for30 min. Then, the tRNA mimic was added to determine if the complex wasformed or inhibited. In this assay a phosphate buffer may be used with1M NaCl to improve the affinity for the two RNA. The stability of thecomplex is concentration dependent so that μM concentrations are usedand the assay is run at 4 degrees C.

The 5′ labeled target RNA sequence (5′-CGGUGUAAAAGC, SEQ ID NO. 5) isbound to a avidin microtiter plate (Roche High Load plates, 96-wellavidin microtiter plates) by adding 150 μl of target solution to eachwell (FIG. 3, step A). The plates are covered and incubated at 37° C.for 1 hour. The plates are then rinsed twice with binding buffer, thesecond rinse is incubated at 37° C. for 5 minutes. The plates are thenrinsed two additional times with binding buffer, covered, and ready foruse.

The test compounds were prepared by thawing solutions of the compoundsto room temperature. Dilutions of the test compounds (1:10 and 1:500)were prepared by dilution in DMSO and shaking for 1 hour.

The assays were performed by adding 98.5 μl of loading buffer (100 mMTris HCl, pH 7.5, 150 mM NaCl and 0.1% Tween 20, pH adjusted from around4.5 to 7.5 with 10 M NaOH) to each well of the plate. Test compoundswere added individually to each well (1.5 μl each), and the plates weremixed for 1 hour (FIG. 3, step B).

Fifty microliters of solution containing the tool tRNA (5′-GCUXUUAYZCUG(SEQ ID NO. 1); where the X, Y, and Z are independently selected frommodified nucleosides mnm5s2U, mcm5s2U, ms2t6A, s2U, ψ, and t6A) was thenadded to each well and the plates were incubated at 4° C. for 1 hourwith shaking (FIG. 3, step C). The reaction mixture was then removed,while the mixture was still cold, and the remaining compound solutionwas also removed.

After removing the remaining solution, reading buffer (50 mM Hepes, pH7.5, 100 mM NaCl, PEG (40 mg/200 ml)) was then added to each well andthe results were read using a plate reader.

As shown in FIG. 3, a positive (+) reaction indicates that the testcompound inhibits binding of the tool tRNA to the target nucleic acid(e.g. the test compound binds to either the tool tRNA, the targetnucleic acid molecule or both the tool tRNA and the target nucleic acidmolecule). A negative (−) reaction indicates that the test compound doesnot inhibit the binding of the tool tRNA to the target nucleic acid(e.g. the test compound does not bind to either the tool tRNA or thetarget nucleic acid).

In the AlphaScreen configuration (FIG. 3) the assay is done in solutionusing the same RNA as the immobilization assay. The donor and acceptorbeads are bound to their respective RNA's. During the screening the RNAsand test drugs/small molecules are incubated together and formation ofthe complex is measured using the AlphaScreen detection conditions.Utilization of the AlphaScreen assay may allow for the assay to be runat a lower RNA concentration at room temperature, and increase thestability of the complex.

Example III. Validation of HIV Screening Assay

The HIV screening assay was validated to confirm that positive andnegative controls would function as expected and to test a smallcompound library to verify that differential inhibition could bedetected. Two validation runs were completed with 4,275 and 4,616compounds, respectively, using 17 plates in each run. There were 3,961compounds in common between the two assays and the statistical analysiswas completed using only these compounds and the positive and negativecontrols. Each plate contained approximately 30 positive and 30 negativecontrols and these controls performed as expected. Differences wereobserved between validation runs when analyzing the luminescence;however, these differences were minimized or eliminated when evaluatingthe percent inhibition by compounds (hits) that were active in bothruns. This assay met the functional requirements based on the results ofthe positive and negative controls.

To evaluate the inhibition exhibited in the screening of this smallcompound library, a cutoff was set at 42.96% inhibition, the averageplus three times standard deviation of compound percent (%) inhibition.Using this cutoff, 34 repeated compounds (hits) were identified. Byusing 99.75% inhibition as the cutoff, the average minus three timesstandard deviation of positive control (Tool+Target) percent (%)inhibition, there is 1 repeated hit. If 29.02%, which is the averageplus three times standard deviation of negative control (Tool) percent(%) Inhibition, is defined as the cutoff, there are 51 repeated hits,out of 3961 compounds analyzed. These results are in line withexpectations when evaluating a small random compound library.

To select compounds for use in a secondary HIV assay to verify that thisassay was capable of identifying HIV specific compounds with biologicalactivity a cutoff was set at greater than 60% inhibition in at least oneof the two validations runs. This resulted in the selection of 29compounds. These compounds were analyzed for anti-HIV activity infreshly harvested PBMC cells. Of the 30 tested compounds, 15 were activeat a concentration of less than 100 ˜M (the highest testedconcentration). Of these 15 compounds, 9 were not toxic to the PBMCcells at the 100 ˜M concentration; thus, an absolute conclusionregarding the differential toxicity to HIV and PBMC cells cannot bedrawn with these 9 compounds. Two other compounds had an antiviral index(inhibited HIV cells and not PBMC cells) greater than 25 which isacceptable.

Example IV: High Throughput Assay on a Large Compound Library

Biochemical HIV-1 tRNA Inhibition Assay:

HIV-1 has evolved to use Human tRNA^(Lys3) as a primer for initiation ofreverse transcription. Therefore, the interaction between tRNA^(Lys3)and viral genomic RNA represents a potential novel target for HIV-1 drugdevelopment. Based on this hypothesis, a biochemical assay to identifyinhibitors of the interaction between Human tRNA^(Lys3) and HIV-1genomic RNA was developed by TRANA Discovery and transferred to SouthernResearch Institute for high-throughput screening. This assay wasdeveloped as a homogeneous amplified luminescent proximity assay usingAlphaScreen™ reagents from PerkinElmer. Based on this assay technology,the AlphaScreen™ luminescent signal serves as a mechanism for detectingthe interaction between RNA molecules that represent Human tRNA^(Lys3)and HIV-1 genomic RNA. The inhibition of the interaction between theseRNA molecules by test compounds is detected as a decrease in theAlphaScreen™ luminescent signal.

Compounds Screened:

A preliminary 15,000 compound screen (NINDS Diversity Set) waspreviously completed and the results were reported through NIAIDcontract N01-AI-70042. For the results described herein, a 101,000compound library that was purchased from ChemBridge for the NIAID TAACFprogram was used. This library was carefully selected for diversity andminimal similarity to the 100,000 compound NINDS library, which is alsoplanned to be screened using the TRANA Discovery Biochemical HIV-1 tRNAInhibition Assay. The compounds were screened at a concentration of 12.5μg/mL (first 3 batches) or 25 μg/mL (fourth and final batch).

Results and Conclusions

Screening Results:

The median Z-value for the 78 assay plates used in the screen was 0.76,with a range from 0.64 to 0.86. Following analysis of the data, 99,303valid screening results were obtained from all four compound batchesscreened. Statistical analysis identified 38.59% inhibition (batches 1-3screened at 12.5 μg/mL) and 44.89% inhibition (batch 4 screened at 25μg/mL) as the cutoffs between inactive and hit compounds. Based on thesestatistical criteria, a total of 315 compounds were identified as hits.There were 202 hits identified from the 75,144 compounds screened incompound batches 1-3 (hit rate of 0.27%). Similarly, there were 113 hitsidentified from the 24,159 compounds screened in compound batch 4 (hitrate of 0.47%). The resulting overall combined hit rate for the screenwas 0.32%. The range of the percent inhibition observed for the 315 hitswas from 38.59% to 99.66%. Overall, the statistical cutoffs and hit rateobserved for this screen are somewhat lower than the values previouslyobserved when screening the NINDS Diversity Set (72.31% and 1.09% forhit cutoff and hit rate, respectively). However, these generaldifferences can be explained by the lower test concentrations and plateformat used for this screen (12.5 and 25 μg/mL; 1536-well plates)compared to the NINDS Diversity Set (40 μg/mL; 384-well plates).

Dose-Response Testing Results:

For initial follow-up testing of the compounds, resupplies of the hitswere purchased from ChemBridge for dose-response testing in the TRANADiscovery biochemical HIV-1 tRNA inhibition assay. Of the 315 hitsidentified in the screen, 309 were available for resupply and wereevaluated in dose-response, in duplicate. 183 hits (59.2%) wereconfirmed to be active in the assay. In addition, 125 hits (40.5%)reached an IC₅₀ value within the concentration range evaluated (0.049-25μg/mL). The observed IC₅₀ values ranged from 0.205 to 24.97 μg/mL.

An additional 200,000 compounds were tested, of which 30,000 compoundscame from an Enamine RDS library, and of which 170,000 compounds camefrom three compound sets (totaling 174,441 compounds), including 1) a99,680 compound library purchased from Enamine 2) a 25,588 compound“Kinase” library purchased from Life Chemicals (LC Kinase), and 3) a49,173 compound ChemBridge CNS subset of the NINDS library. Based on theresults of the high throughput screening using these libraries, 1,117compounds were identified as hits. These hits were cherry-picked fromthe compound library plates for follow-up testing in dose-response inthe TRANA Discovery Biochemical HIV-1 tRNA Inhibition Assay as well asin a cytotoxicity assay using the human monocyte cell line, THP-1. Basedon the dose-response testing of hits, 93 compounds that achieved an IC50value of less than 25 μg/mL in the TRANA Discovery HIV-1 tRNAbiochemical inhibition assay and that were not toxic in the THP-1cytotoxicity assay were identified for additional follow-up testing.Eighty-eight (88) of these compounds were available for re-supply fromthe library suppliers and purchased in larger quantities for testingagainst HIV-1IIIB replication in a CEM-SS cytoprotection assay using a100 μM high-test concentration. AZT was included in the CEM-SScytoprotection assay as a positive control antiviral compound.

The active compounds are listed below, with those compounds showing someactivity marked with a “+,” those with intermediate activity marked witha “++,” and those with the highest activity marked with a “+++.”

-   ethyl 5-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoate-   Activity: +++-   1-(1-pyrrolidinyl)anthra-9,10-quinone-   1-Pyrrolidin-1-yl-anthraquinone-   ++-   ethyl (3-oxo-4,5-diphenyl-1,3-dihydro-2H-pyrrol-2-ylidene)acetate-   Activity: ++-   1-methyl-4-(1-piperidinyl)anthra-9,10-quinone-   1-Methyl-4-piperidin-1-yl-anthraquinone-   Activity: ++-   2-[(3-methylphenyl)amino]-3-(4-morpholinyl)naphthoquinone-   Activity: ++-   2-[(3-fluorophenyl)amino]-3-(1-pyrrolidinyl)naphthoquinone-   Activity: ++-   N-(4-ethoxy-8-methyl-2-quinazolinyl)guanidine-   Activity: ++-   2-[(2-methylphenyl)amino]-3-(4-morpholinyl)naphthoquinone-   Activity: ++-   2-[(3-methylphenyl)amino]-3-(1-pyrrolidinyl)naphthoquinone-   Activity: ++-   2-[(3-methoxyphenyl)amino]-3-(1-piperidinyl)naphthoquinone-   Activity: ++-   2-[(3-methoxyphenyl)amino]-3-(4-morpholinyl)naphthoquinone-   Activity: ++-   1-(dimethylamino)anthra-9,10-quinone-   1-Dimethylamino-anthraquinone-   Activity: ++-   2-[(3-chlorophenyl)amino]-3-(4-morpholinyl)naphthoquinone-   Activity: ++-   ethyl    4-{[3-(4-morpholinyl)-1,4-dioxo-1,4-dihydro-2-naphthalenyl]amino}benzoate-   Activity: ++-   methyl    4-{[1,4-dioxo-3-(1-pyrrolidinyl)-1,4-dihydro-2-naphthalenyl]amino}benzoate-   Activity: ++-   1-(4-hydroxyphenyl)-1-propanone (2-nitrophenyl)hydrazone-   Activity: ++-   4-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]phenol-   Activity: ++-   4,4,8-trimethyl-1-{[2-(4-nitrophenyl)-2-oxoethyl]thio}-4,5-dihydro[1,2]dithiolo[3,4-c]quinolin-2-ium    chloride-   Activity: ++-   2-[(4-acetylphenyl)amino]-3-(1-pyrrolidinyl)naphthoquinone-   Activity: ++-   3,4-dihydroxybenzaldehyde (2-nitrophenyl)hydrazone-   Activity: ++-   2,6-dimethyl-4-[(4-methylphenyl)amino]phenol-   Activity: ++-   5-(3,4-dihydroxybenzylidene)-3-(3-fluorophenyl)-2-thioxo-1,3-thiazolidin-4-one-   Activity: ++-   methyl    9,10-dioxo-4-(1-piperidinyl)-9,10-dihydro-1-anthracenecarboxylate-   Activity: ++-   2-[(3-methoxyphenyl)amino]-3-(1-pyrrolidinyl)naphthoquinone-   Activity: ++-   2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]phenol-   Activity: ++-   2-chloro-3-(dimethylamino)naphthoquinone-   Activity: ++-   methyl    4-{[3-(4-morpholinyl)-1,4-dioxo-1,4-dihydro-2-naphthalenyl]amino}benzoate-   Activity: ++-   1-(3-methylphenyl)-5-[(1-methyl-1H-pyrrol-2-yl)methylene]-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione-   Activity: ++-   8-ethoxy-4,4-dimethyl-1-(methylthio)-4,5-dihydro[1,2]dithiolo[3,4-c]quinolin-2-ium    chloride-   Activity: ++-   2-[(3,5-difluorophenyl)amino]-3-(4-morpholinyl)naphthoquinone-   Activity: ++-   2-(dimethylamino)-3-[(3-methylphenyl)amino]naphthoquinone-   Activity: ++-   5-(2,6-dichloro-3-nitrobenzylidene)-3-methyl-2-thioxo-1,3-thiazolidin-4-one-   Activity: ++-   5-(3,4-dihydroxybenzylidene)-3-ethyl-2-thioxo-1,3-thiazolidin-4-one-   Activity: +-   N-(4-ethoxyphenyl)-7-nitro-2,1,3-benzoxadiazol-4-amine-   Activity: +-   2-[(3,4-difluorophenyl)amino]-3-(4-morpholinyl)naphthoquinone-   Activity: +-   N,N-dimethyl-N′-(7-methyl-2,3-dihydro-1H-cyclopenta[b]quinolin-9-yl)-1,4-benzenediamine-   Activity: +-   1-(4-chlorophenyl)-5-(2-furylmethylene)-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione-   Activity: +-   5-(2-furylmethylene)-1-(1-naphthyl)-2,4,6(1H,3H,5H)-pyrimidinetrione-   Activity: +-   N-[4-(dimethylamino)benzyl]-N,N′,N′-triethyl-1,2-ethanediamine-   Activity: +-   3-allyl-5-(3,5-dichloro-2-hydroxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one-   Activity: +-   2-chloro-3-(isopropylamino)naphthoquinone-   Activity: +-   2-[(3-hydroxyphenyl)amino]-3-(1-piperidinyl)naphthoquinone-   Activity: +-   N,N-diethyl-N′-[4-(2-pyridinyl)-1,3-thiazol-2-yl]-1,4-benzenediamine    hydrobromide-   Activity: +-   N-(7-methoxy-4-methyl-2-quinazolinyl)guanidine-   Activity: +-   3-nitrobenzaldehyde 1,2-ethanediyl(methylhydrazone)-   Activity: +-   3-amino-5-(3,4-dihydroxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one-   Activity: +-   2-(benzylamino)-3-chloronaphthoquinone-   Activity: +-   1-amino-2-chloro-4-(1-pyrrolidinyl)anthra-9,10-quinone-   1-Amino-2-chloro-4-pyrrolidin-1-yl-anthraquinone-   Activity: +-   N-({[4-(diethylamino)phenyl]amino}carbonothioyl)-3-isopropoxybenzamide-   Activity: +-   5-acetyl-8-ethoxy-4,4-dimethyl-4,5-dihydro-1H-[1,2]dithiolo[3,4-c]quinoline-1-thione-   1-(8-Ethoxy-4,4-dimethyl-1-thioxo-1,3a,4,9b-tetrahydro-2,3-dithia-5-aza-cyclopenta[a]naphthalen-5-yl)-ethanone-   Activity: +-   1-(3-chlorophenyl)-5-(2-furylmethylene)-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione-   Activity: +-   1-(4-chlorophenyl)-5-[(1-methyl-1H-pyrrol-2-yl)methylene]-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione-   Activity: +-   1,3-benzodioxol-5-yl(3-methylbenzyl)amine-   Activity: +-   (4-{3-[4-(2-methoxyphenyl)-1-piperazinyl]-1-propen-1-yl}phenyl)dimethylamine-   Activity: +-   ethyl    2-(7-bromo-3,4-dioxo-3,4-dihydro-1-naphthalenyl)-3-oxo-3-phenylpropanoate-   Activity: +-   5-acetyl-4,4,8-trimethyl-4,5-dihydro-1H[1,2]dithiolo[3,4-c]quinoline-1-thione-   1-(4,4,8-Trimethyl-1-thioxo-1,3a,4,9b-tetrahydro-2,3-dithia-5-aza-cyclopenta[a]naphthalen-5-yl)-ethanone-   Activity: +-   1-(2-furyl)ethanone (2-nitrophenyl)hydrazone-   Activity: +-   5-acetyl-8-methoxy-4,4-dimethyl-4,5-dihydro-1H-[1,2]dithiolo[3,4-c]quinoline-1-thione-   1-(8-Methoxy-4,4-dimethyl-1-thioxo-1,3a,4,9b-tetrahydro-2,3-dithia-5-aza-cyclopenta[a]naphthalen-5-yl)-ethanone-   Activity: +-   2-[(4,7-dimethyl-2-quinazolinyl)amino]-6-methyl-4-pyrimidinol-   Activity: +-   2-amino-3-[(2-methylphenyl)amino]naphthoquinone-   Activity: +-   1-(5-chloro-2-nitrophenyl)-4-(4-methoxyphenyl)piperazine-   Activity: +-   1-(4-chlorophenyl)-5-[(5-nitro-2-furyl)methylene]-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione-   Activity: +-   5-(3,4-dihydroxybenzylidene)-3-phenyl-2-thioxo-1,3-thiazolidin-4-one-   Activity: +-   3-butoxy-N-({[4-(dimethylamino)phenyl]amino}carbonothioyl)benzamide-   Activity: +-   2-bromo-N-[5-(3,4-dihydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]benzamide-   Activity: +-   5-methyl-2-furaldehyde (2-nitrophenyl)hydrazone-   Activity: +-   N-(4,8-dimethyl-2-quinazolinyl)guanidine-   Activity: +-   2-[(2-hydroxyethyl)amino]-3-[(3-methylphenyl)amino]naphthoquinone-   Activity: +-   1-[(2,2,6,6-tetramethyl-4-piperidinyl)amino]anthra-9,10-quinone-   1-[1-(2,2-Dimethyl-propyl)-3,3-dimethyl-butylamino]-anthraquinone-   Activity: +-   6-(5-methyl-2-oxo-2,3-dihydro-1H-imidazol-4-yl)-6-oxohexanoic acid-   Activity: +++-   ethyl 6-(5-methyl-2-oxo-2,3-dihydro-1H-imidazol-4-yl)-6-oxohexanoate-   Activity: ++

These compounds have been prioritized based on their IC₅₀ values andwere subjected to dose-response testing against HIV-1_(IIIB) replicationin a CEM-SS cytoprotection assay, as shown below in Example V.

Example V: High Throughput In Vivo Assay—Efficacy Evaluation in CEM-SSCells

a. Anti-HIV-1 Cytoprotection Assay

Cell Preparation—CEM-SS cells were passaged in T-75 flasks prior to usein the antiviral assay. On the day preceding the assay, the cells weresplit 1:2 to assure they were in an exponential growth phase at the timeof infection. Total cell and viability quantification was performedusing a hemacytometer and trypan blue exclusion. Cell viability wasgreater than 95% for the cells to be utilized in the assay. The cellswere resuspended at 5×10⁴ cells/mL in tissue culture medium and added tothe drug-containing microtiter plates in a volume of 50 μL.

Virus Preparation—The virus used for this test was the lymphocytropicvirus strains HIV-1IIIB The virus was obtained from the NIH AIDSResearch and Reference Reagent Program and was grown in CEM-SS cells forthe production of stock virus pools. For each assay, a pre-titeredaliquot of virus was removed from the freezer (−80° C.) and allowed tothaw slowly to room temperature in a biological safety cabinet. Thevirus was resuspended and diluted into tissue culture medium such thatthe amount of virus added to each well in a volume of 50 μL was theamount determined to give between 85 to 95% cell killing at 6 dayspost-infection. TCID₅₀ calculations by endpoint titration in CEM-SScells indicated that the multiplicity of infection of these assays wasapproximately 0.01.

The table below shows the plate format used in the assay:

1 2 3 4 5 6 7 8 9 10 11 12 A Reagent Background Control Wells PlasticBackground Control Wells (Media plus MTS, no cells) (Media only, nocells) B Tox 1 Cell Drug 1 Low-Test Tox 1 Tox 2 Drug 2 Low-Test Cell Tox2 0.32 μM  Control 0.32 μM  0.32 μM  0.32 μM  0.32 μM  Control 0.32 μM C Tox 1 Drug 1 Tox 1 Tox 2 Drug 2 Tox 2 1.0 μM 1.0 μM  1.0 μM 1.0 μM 1.0μM  1.0 μM D Tox 1 Drug 1 Tox 1 Tox 2 Drug 2 Tox 2 3.2 μM 3.2 μM  3.2 μM3.2 μM 3.2 μM  3.2 μM E Tox 1 Virus Drug 1 Tox 1 Tox 2 Drug 2 Virus Tox2  10 μM Control 10 μM  10 μM  10 μM 10 μM Control  10 μM F Tox 1 Drug 1Tox 1 Tox 2 Drug 2 Tox 2  32 μM 32 μM  32 μM  32 μM 32 μM  32 μM G Tox 1Drug 1 High-Test Tox 1 Tox 2 Drug 2 High-Test Tox 2 100 μM  100 μM  100μM  100 μM  100 μM  100 μM  H Color 1 Color 1 Color 1 Color 1 Color 1Color 1 Color 2 Color 2 Color 2 Color 2 Color 2 Color 2 100 μM  32 μM 10μM 3.2 μM 1.0 μM 0.32 μM  100 μM  32 μM 10 μM 3.2 μM 1.0 μM 0.32 μM Cells labeled as “Drug 1” or “Drug 2” = Cells + Virus + Drug (exampledilution scheme indicated) Cells labeled as “Tox 1” or “Tox 2” = Cells +Drug 1 or Drug 2, respectively (toxicity tested in duplicate) Cellslabeled as “Color 1” or “Color 2” = Media + Drug 1 or Drug 2,respectively (colorimetric background, no cells)Plate Format—The format of the test plate is shown in the table above.Each plate contains cell control wells (cells only), virus control wells(cells plus virus), drug cytotoxicity wells (cells plus drug only), drugcolorimetric control wells (drug only) as well as experimental wells(drug plus cells plus virus). As shown in the table above, 100 μM wasused as a representative high-test concentration. Samples were evaluatedfor antiviral efficacy with triplicate measurements using 6concentrations at half-log dilutions in order to determine IC₅₀ valuesand with duplicate measurements to determine cellular toxicity, ifdetectable.

b. MTS Staining for Cell Viability

At assay termination, the assay plates were stained with the solubletetrazolium-based dye MTS (CellTiter 96 Reagent, Promega) to determinecell viability and quantify compound toxicity. MTS is metabolized by themitochondrial enzymes of metabolically active cells to yield a solubleformazan product, allowing the rapid quantitative analysis of cellviability and compound cytotoxicity. This reagent is a stable, singlesolution that does not require preparation before use. At termination ofthe assay, 20-25 μL of MTS reagent is added per well and the microtiterplates are then incubated for 4-6 hrs at 37° C., 5% CO₂ to assess cellviability. Adhesive plate sealers were used in place of the lids, thesealed plate was inverted several times to mix the soluble formazanproduct and the plate was read spectrophotometrically at 490/650 nm witha Molecular Devices Vmax plate reader.

c. Data Analysis

Using a proprietary computer program % CPE Reduction, % Cell Viability,compound concentrations resulting in the inhibition of virus replicationby 25%, 50%, and 95% (IC₂₅, IC₅₀, and IC₉₅ values), compoundconcentrations resulting in 25%, 50%, and 95% cytotoxicity (TC₂₅, TC₅₀,and TC₉₅) and Antiviral Indices (=TC₂₅/IC₂₅, TC₅₀/IC₅₀, or TC₉₅/IC₉₅)were calculated and the graphical results summary was displayed. AZT wasevaluated in parallel as a relevant positive control compound in theanti HIV assays.

Results

The results from the testing of 88 lead compounds identified in in vitroassays against HIV-1IIIB replication in CEM-SS cells are summarizedbelow. AZT was used as a positive control. Those compounds listed belowhad bioactivity in the assay.

Compound 82 has the structure shown below:

Compound 576 has the structure shown below:

Compound 374 has the structure shown below:

Compound 257 has the structure shown below:

Compound 259 has the structure shown below:

Compound 110 has the structure shown below:

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A pharmaceutical composition comprising acompound of the following formula:

wherein: R₄, R₅, R₆, R₇, R₁₅, R₁₆, and R₁₇ are, independently, the sameor different, and are selected from the group consisting of hydrogen,C₁-C₆ alkyl, piperidinyl, phenyl, naphthyl, C₂₋₆ alkenyl, and halo, anda pharmaceutically-acceptable carrier, and further comprising anadditional antiviral agent selected from the group consisting of entryinhibitors, integrase inhibitors, reverse transcriptase inhibitors, andimmune-based therapeutic agents.
 2. A pharmaceutical compositioncomprising a compound of Formula H:

Formula H wherein R⁴, R⁵, R⁶, R⁷, R¹⁵, R¹⁶, and R¹⁷ are, independently,the same or different, and are selected from the group consisting ofhydrogen, C₁-C₆ alkyl, piperidinyl, phenyl, naphthyl, C₂-C₆ alkenyl, andhalo, and further comprising an additional antiviral agent selected fromthe group consisting of entry inhibitors, integrase inhibitors, reversetranscriptase inhibitors, and immune-based therapeutic agents.
 3. Thecomposition of claim 1, wherein R₄, R₅, R₆, R₇, R₁₅, R₁₆, and R₁₇ are,independently, the same or different, and are selected from the groupconsisting of hydrogen, C₁-C₆ alkyl, and halo.
 4. The composition ofclaim 3, wherein the C₁-C₆ alkyl moiety is methyl.
 5. The composition ofclaim 2, wherein R₄, R₅, R₆, R₇, R₁₅, R₁₆, and R₁₇ are, independently,the same or different, and are selected from the group consisting ofhydrogen, C₁-C₆ alkyl, and halo.
 6. The composition of claim 5, whereinthe C₁-C₆ alkyl is methyl.
 7. A method of treating or preventing aretroviral infection, comprising administering a composition of claim 2.8. The method of claim 7, further comprising the co-administration of asecond antiretroviral compound.
 9. The method of claim 8, wherein thesecond antiretroviral agent is selected from the group consisting ofNRTIs, NNRTIs, VAP anti-idiotypic antibodies, CD4 and CCR5 receptorinhibitors, entry inhibitors, antisense oligonucleotides, ribozymes,protease inhibitors, neuraminidase inhibitors, tyrosine kinaseinhibitors, PI-3 kinase inhibitors, and Interferons.
 10. The method ofany of claim 7, wherein the retrovirus is selected from the groupconsisting of Feline Immunodeficiency Virus (FIV), SimianImmunodeficiency Virus (SIV), Avian Leucosis Virus, Feline LeukemiaVirus, Walleye Dermal Sarcoma Virus, Human T-Lymphotropic Virus, andHuman Immunodeficiency Viruses (HIV).
 11. The method of any of claim 7,wherein the retrovirus is HIV.
 12. The method of claim 11, wherein theHIV is selected from the group consisting of HIV-I, HIV-II, HIV-III andmutated versions thereof.